Transgenic non-human animals expressing a gdf-11 dominant negative polypeptide, and methods of making and using same

ABSTRACT

A transgenic non-human animal of the species selected from the group consisting of avian, bovine, ovine and porcine having a transgene which results in disrupting the production of and/or activity of growth differentiation factor-11 (GDF-11) chromosomally integrated into the germ cells of the animal is disclosed. Also disclosed are methods for making such animals, and methods of treating animals, including humans, with antibodies or antisense directed to GDF-11. The animals so treated are characterized by increased muscle tissue and bone tissue.

[0001] This application is a continuation-in-part application of U.S.application Ser. No. 09/019,901, filed Feb. 6, 1998 which is acontinuation-in-part of U.S. application Ser. No. 08/795,671, filed Feb.6, 1997 and U.S. application Ser. No. 08//706,958, filed Sep. 3, 1996,which is a continuation of U.S. application Ser. No. 08/272,763, filedJul. 8, 1994 now abandoned.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The invention relates generally to growth factors andspecifically to a new member of the transforming growth factor beta(TGF-β) superfamily, which is denoted, growth differentiation factor-11(GDF-11) and methods of use for modulating muscle cell, bone, kidney andadipose tissue growth.

[0004] 2. Description of Related Art

[0005] The transforming growth factor β (TGF-β) superfamily encompassesa group of structurally-related proteins which affect a wide range ofdifferentiation processes during embryonic development. The familyincludes, Mullerian inhibiting substance (MIS), which is required fornormal male sex development (Behringer, et al., Nature, 345:167, 1990),Drosophila decapentaplegic (DPP) gene product, which is required fordorsal-ventral axis formation and morphogenesis of the imaginal disks(Padgett, et al., Nature, 325:81-84, 1987), the Xenopus Vg-1 geneproduct, which localizes to the vegetal pole of eggs ((Weeks, et al.,Cell, 51:861-867, 1987), the activins (Mason, et al., Biochem, Biophys.Res. Commun., 135:957-964, 1986), which can induce the formation ofmesoderm and anterior structures in Xenopus embryos (Thomsen, et al.,Cell, 63:485, 1990), and the bone morphogenetic proteins (BMPs,osteogenin, OP-1) which can induce de novo cartilage and bone formation(Sampath, et al., J. Biol. Chem., 265:13198, 1990). The TGF-βs caninfluence a variety of differentiation processes, includingadipogenesis, myogenesis, chondrogenesis, hematopolesis, and epithelialcell differentiation (for review, see Massague, Cell 49:437, 1987).

[0006] The proteins of the TGF-β family are initially synthesized as alarge precursor protein which subsequently undergoes proteolyticcleavage at a cluster of basic residues approximately 110-140 aminoacids from the C-terminus. The C-terminal regions, or mature regions, ofthe proteins are all structurally related and the different familymembers can be classified into distinct subgroups based on the extent oftheir homology. Although the homologies within particular subgroupsrange from 70% to 90% amino acid sequence identity, the homologiesbetween subgroups are significantly lower, generally ranging from only20% to 50%. In each case, the active species appears to be adisulfide-linked dimer of C-terminal fragments. Studies have shown thatwhen the pro-region of a member of the TGF-β family is coexpressed witha mature region of another member of the TGF-β family, intracellulardimerization and secretion of biologically active homodimers occur(Gray, A. et al., Science, 247:1328, 1990). Additional studies byHammonds, et al., (Molec. Endocrin. 5:149, 1991) showed that the use ofthe BMP-2 pro-region combined with the BMP-4 mature region led todramatically improved expression of mature BMP-4. For most of the familymembers that have been studied, the homodimeric species has been foundto be biologically active, but for other family members, like theinhibins (Ling, et al., Nature, 321:779, 1986) and the TGF-βs (Cheifetz,et al., Cell, 48:409, 1987), heterodimers have also been detected, andthese appear to have different biological properties than the respectivehomodimers.

[0007] In addition, it is desirable to produce livestock and gameanimals, such as cows, sheep, pigs, chicken and turkey, fish which arerelatively high in musculature and protein, and low in fat content. Manydrug and diet regimens exist which may help increase muscle and proteincontent and lower undesirably high fat and/or cholesterol levels, butsuch treatment is generally administered after the fact, and is begunonly after significant damage has occurred to the vasculature.Accordingly, it would be desirable to produce animals which aregenetically predisposed to having higher muscle content, without anyancillary increase in fat levels.

[0008] The food industry has put much effort into increasing the amountof muscle and protein in foodstuffs. This quest is relatively simple inthe manufacture of synthetic foodstuffs, but has been met with limitedsuccess in the preparation of animal foodstuffs. Attempts have beenmade, for example, to lower cholesterol levels in beef and poultryproducts by including cholesterol-lowering drugs in animal feed (seee.g. Elkin and Rogler, J. Agric. Food Chem. 1990, 38, 1635-1641).However, there remains a need for more effective methods of increasingmuscle and reducing fat and cholesterol levels in animal food products.

SUMMARY OF THE INVENTION

[0009] The present invention provides a cell growth and differentiationfactor, GDF-11, a polynucleotide sequence which encodes the factor, andantibodies which are immunoreactive with the factor. This factor appearsto relate to various cell proliferative disorders, especially thoseinvolving muscle, nerve, bone, kidney and adipose tissue.

[0010] In one embodiment, the invention provides a method for detectinga cell proliferative disorder of muscle, nerve, bone, kidney or fatorigin and which is associated with GDF-11. In another embodiment, theinvention provides a method for treating a cell proliferative disorderby suppressing or enhancing GDF-11 activity.

[0011] In another embodiment, the subject invention provides non-humantransgenic animals which are useful as a source of food products withhigh muscle, bone and protein content, and reduced fat and cholesterolcontent. The animals have been altered chromosomally in their germ cellsand somatic cells so that the production of GDF-11 is produced inreduced amounts, or is completely disrupted, resulting in animals withdecreased levels of GDF-11 in their system and higher than normal levelsof muscle tissue and bone tissue, such as ribs, preferably withoutincreased fat and/or cholesterol levels. Accordingly, the presentinvention also includes food products provided by the animals. Such foodproducts have increased nutritional value because of the increase inmuscle tissue and bone tissue to which the muscle attaches. Thetransgenic non-human animals of the invention include bovine, porcine,ovine and avian animals, for example.

[0012] The subject invention also provides a method of producing animalfood products having increased muscle content. The method includesmodifying the genetic makeup of the germ cells of a pronuclear embryo ofthe animal, implanting the embryo into the oviduct of a pseudopregnantfemale thereby allowing the embryo to mature to full term progeny,testing the progeny for presence of the transgene to identifytransgene-positive progeny, cross-breeding transgene-positive progeny toobtain further transgene-positive progeny and processing the progeny toobtain foodstuff. The modification of the germ cell comprises alteringthe genetic composition so as to disrupt or reduce the expression of thenaturally occurring gene encoding for production of GDF-11 protein. In aparticular embodiment, the transgene comprises antisense polynucleotidesequences to the GDF-11 protein. Alternatively, the transgene maycomprise a non-functional sequence which replaces or intervenes in thenative GDF-11 gene.

[0013] The subject invention also provides a method of producing animalfood products having increased bone content. The method includesmodifying the genetic makeup of the germ cells of a pronuclear embryo ofthe animal, implanting the embryo into the oviduct of a pseudopregnantfemale thereby allowing the embryo to mature to full term progeny,testing the progeny for presence of the transgene to identifytransgene-positive progeny, cross-breeding transgene-positive progeny toobtain further transgene-positive progeny and processing the progeny toobtain foodstuff. The modification of the germ cell comprises alteringthe genetic composition so as to disrupt or reduce the expression of thenaturally occurring gene encoding for production of GDF-11 protein. In aparticular embodiment, the transgene comprises antisense polynucleotidesequences to the GDF-11 protein. Alternatively, the transgene maycomprise a non-functional sequence which replaces or intervenes in thenative GDF-11 gene.

[0014] The subject invention also provides a method of producing avianfood products having improved muscle and/or bone content. The methodincludes modifying the genetic makeup of the germ cells of a pronuclearembryo of the avian animal, implanting the embryo into the oviduct of apseudopregnant female into an embryo of a chicken, culturing the embryounder conditions whereby progeny are hatched, testing the progeny forpresence of the genetic alteration to identify transgene-positiveprogeny, cross-breeding transgene-positive progeny and processing theprogeny to obtain foodstuff.

[0015] The invention also provides a method for treating a muscle, bone,kidney or adipose tissue disorder in a subject. The method includesadministering a therapeutically effective amount of a GDF-11 agent tothe subject, thereby affecting growth of muscle, bone, kidney or adiposetissue. The GDF-11 agent may include an antibody, a GDF-11 antisensemolecule or a dominant negative polypeptide, for example. In one aspect,a method for inhibiting the growth regulating actions of GDF-11 bycontacting an anti-GDF-11 monoclonal antibody, a GDF-11 antisensemolecule or a dominant negative polypeptide (or polynucleotide encodinga dominant negative polypeptide) with fetal or adult muscle cells orprogenitor cells is included. These agents can be administered to apatient suffering from a disorder such as muscle wasting disease,neuromuscular disorder, muscle atrophy, obesity or other adipocyte celldisorders, and aging, for example. In another aspect of the invention,the agent may be an agonist of GDF-11 activity.

[0016] The invention also provides a method for identifying a compoundthat affects GDF-11 activity or gene expression including incubating thecompound with GDF-11 polypeptide, or with a recombinant cell expressingGDF-11 under conditions sufficient to allow the compounds to interactand determining the effect of the compound on GDF-11 activity orexpression.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017]FIG. 1 shows the nucleotide and predicted amino acid sequences ofmurine (FIG. 1a) and human (FIG. 1b) GDF-11. The putative proteolyticprocessing sites are shown by the shaded boxes. In the human sequence,the potential N-linked glycosylation signal is shown by the open box,and the consensus polyadenylation signal is underlined; the poly A tailis not shown.

[0018]FIG. 2 shows Northern blots of RNA prepared from adult (FIG. 2a)or fetal and neonatal (FIG. 2b) tissues probed with a murine GDF-11probe.

[0019]FIG. 3 shows amino acid homologies among different members of theTGF-β superfamily. Numbers represent percent amino acid identitiesbetween each pair calculated from the first conserved cysteine to theC-terminus. Boxes represent homologies among highly-related memberswithin particular subgroups.

[0020]FIG. 4a shows an alignment of the predicted amino acid sequencesof human GDF-11 (top lines) with human GDF-8 (bottom lines). Verticallines indicate identities. Dots represent gaps introduced in order tomaximize the alignment. Numbers represent amino acid positions relativeto the N-terminus. The putative proteolytic processing sites are shownby the open box. The conserved cysteine residues on the C-terminalregion are shown by the shaded boxes.

[0021]FIG. 4b shows the predicted amino acid sequences of murine andhuman GDF-11 aligned with murine (McPherron et al., 1997) and human(McPherron and Lee, 1997) myostatin (MSTN). Shaded boxes represent aminoacid homology with the murine and human GDF-11 sequences. Amino acidsare numbered relative to the human GDF-11 sequence. The predictedproteolytic processing sites are located at amino acids 295-298.

[0022]FIG. 5 shows the expression of GDF-11 in mammalian cells.Conditioned medium prepared from Chinese hamster ovary cells transfectedwith a hybrid GDF-8/GDF-11 gene (see text) cloned into the MSXNDexpression vector in either the antisense (lane 1) or sense (lane 2)orientation was dialyzed, lyophilized, and subjected to Western analysisusing antibodies directed against the C-terminal portion of GDF-8protein. Arrows at right indicate the putative unprocessed(pro-GDF-8/GDF-11) or processed GDF-11 proteins. Numbers at leftindicate mobilities of molecular weight standards.

[0023]FIG. 6 shows the chromosomal mapping of human GDF-11. DNA samplesprepared from human/rodent somatic cell lines were subjected to PCR,electrophoresed on agarose gels, blotted, and probed. The humanchromosome contained in each of the hybrid cell lines is identified atthe top of each of the first 24 lanes (1-22, X, and Y). In the lanesdesignated CHO, M, and H, the starting DNA template was total genomicDNA from hamster, mouse, and human sources, respectively. In the lanemarked B1, no template DNA was used. Numbers at left indicate themobilities of DNA standards.

[0024]FIG. 7 shows the FISH localization of GDF-11. Metaphasechromosomes derived from peripheral blood lymphocytes were hybridizedwith digoxigenin-labeled human GDF-11 probe (a) or a mixture of humanGDF-11 genomic and chromosome 12-specific centromere probes (b) andanalyzed as described in the text. A schematic showing the location ofGDF-11 at position 12q13 is shown in panel (c).

[0025]FIG. 8 shows a genomic Southern analysis of DNA isolated fromdifferent species.

[0026]FIG. 9 shows the construction of GDF-11 null mice by homologoustargeting. a) is a map of the GDF-11 locus (top line) and targetingconstruct (second line). The black and stippled boxes represent codingsequences for the pro-and C-terminal regions, respectively. Thetargeting construct contains a total of 11 kb of homology with theGDF-11 gene. A probe derived from the region upstream of the 3′ homologyfragment and downstream of the first EcoRI site shown hybridizes to a6.5 kb EcoR1 fragment in the GDF-11 gene and a 4.8 kb fragment in ahomologously targeted gene. Abbreviations: X, Xba1; E, EcoR1. b)Geneomic Southern of DNA prepared from F1 heterozygous mutant mice(lanes 1 and 2) and offspring derived from a mating of these mice (lanes3-12).

[0027]FIG. 10 shows kidney abnormalities in GDF-11 knockout mice.Kidneys of newborn animals were examined and classified according to thenumber of normal sized or small kidneys as shown at the top. Numbers inthe table indicate number of animals falling into each classificationaccording to genotype.

[0028]FIG. 11 shows homeotic transformations in GDF-11 mutant mice. a)Newborn pups with missing (first and second from left) and normallooking tails. b-j) Skeleton preparations for newborn wild-type (b, e,h), heterozygous (c, f, i) and homozygous (d, g, j) mutant mice. Wholeskeleton preparations (b-d), vertebral columns (e-g), vertebrosternalribs (h-j) showing transformations and defects in homozygous andheterozygous mutant mice. Numbers indicate thoracic segments.

[0029]FIG. 12 is a table summarizing the anterior transformations inwild-type, heterozygous and homozygous GDF-11 mice.

DETAILED DESCRIPTION OF THE INVENTION

[0030] The present invention provides a growth and differentiationfactor, GDF-11, and a polynucleotide sequence encoding GDF-11. GDF-11 isexpressed at highest levels in muscle, brain, uterus, spleen, and thymusand at lower levels in other tissues.

[0031] The TGF-β superfamily consists of multifunctional polypeptidesthat control proliferation, differentiation, and other functions in manycell types. Many of the peptides have regulatory, both positive andnegative, effects on other peptide growth factors. The structuralhomology between the GDF-11 protein of this invention and the members ofthe TGF-β family, indicates that GDF-11 is a new member of the family ofgrowth and differentiation factors. Based on the known activities ofmany of the other members, it can be expected that GDF-11 will alsopossess biological activities that will make it useful as a diagnosticand therapeutic reagent.

[0032] Certain members of this superfamily have expression patterns orpossess activities that relate to the function of the nervous system.For example, one family member, namely GDNF, has been shown to be apotent neurotrophic factor that can promote the survival of dopaminergicneurons (Lin, et al., Science, 260:1130). Another family member, namelydorsalin-1, is capable of promoting the differentiation of neural crestcells (Basler, et al., Cell, 73:687, 1993). The inhibins and activinshave been shown to be expressed in the brain (Meunier, et al., Proc.Nat'l. Acad. Sci., USA, 85:247, 1988; Sawchenko, et al., Nature,334:615, 1988), and activin has been shown to be capable of functioningas a nerve cell survival molecule (Schubert, et al., Nature, 344:868,1990). Another family member, namely GDF-1, is nervous system-specificin its expression pattern (Lee, Proc. Nat'l. Acad. Sci., USA, 88:4250,1991), and certain other family members, such as Vgr-1 (Lyons, et al.,Proc. Nat'l. Acad. Sci., USA, 86:4554, 1989; Jones, et al., Development,111:581, 1991), OP-1 (Ozkaynak, et al., J. Biol. Chem., 267:25220,1992), and BMP4 (Jones, et al., Development, 111:531, 1991), are alsoknown to be expressed in the nervous system. The expression of GDF-11 inbrain and muscle suggests that GDF-11 may also possess activities thatrelate to the function of the nervous system. In particular, it isknown, for example, that skeletal muscle produces a factor or factorsthat promote the survival of motor neurons (Brown, Trends Neurosci.,7:10, 1984). The known neurotrophic activities of other members of thisfamily and the expression of GDF-11 in muscle suggest that one activityof GDF-11 may be as a trophic factor for motor neurons; indeed, GDF-11is highly related to GDF-8, which is virtually muscle-specific in itsexpression pattern. Alternatively, GDF-11 may have neurotrophicactivities for other neuronal populations. Hence, GDF-11 may have invitro and in vivo applications in the treatment of neurodegenerativediseases, such as amyotrophic lateral sclerosis, or in maintaining cellsor tissues in culture prior to transplantation.

[0033] GDF-11 may also have applications in treating disease processesinvolving the musculoskeletal system, such as in musculodegenerativediseases, osteoporosis or in tissue repair due to trauma. In thisregard, many other members of the TGF-β family are also importantmediators of tissue repair. TGF-β has been shown to have marked effectson the formation of collagen and to cause a striking angiogenic responsein the newborn mouse (Roberts, et al., Proc. Natl. Acad. Sci., USA83:4167, 1986). TGF-β has also been shown to inhibit the differentiationof myoblasts in culture (Massague, et al., Proc. Natl. Acad. Sci., USA83:8206, 1986). Moreover, because myoblast cells may be used as avehicle for delivering genes to muscle for gene therapy, the propertiesof GDF-11 could be exploited for maintaining cells prior totransplantation or for enhancing the efficiency of the fusion process.GDF-11 may also have applications in treating disease processesinvolving the kidney or in kidney repair due to trauma.

[0034] GDF-11 may also have applications in the treatment of immunologicdisorders. In particular, TGF-β has been shown to have a wide range ofimmunoregulatory activities, including potent suppressive effects on Band T cell proliferation and function (for review, see Palladino, etal., Ann. N.Y. Acad. Sci., 593:181, 1990). The expression of GDF-11 inspleen and thymus suggests that GDF-11 may possess similar activitiesand therefore, may be used as an anti-inflammatory agent or as atreatment for disorders related to abnormal proliferation or function oflymphocytes.

[0035] The animals contemplated for use in the practice of the subjectinvention are those animals generally regarded as useful for theprocessing of food stuffs, i.e. avian such as meat bred and egg layingchicken and turkey, ovine such as lamb, bovine such as beef cattle andmilk cows, piscine and porcine. For purposes of the subject invention,these animals are referred to as “transgenic” when such animal has had aheterologous DNA sequence, or one or more additional DNA sequencesnormally endogenous to the animal (collectively referred to herein as“transgenes”) chromosomally integrated into the germ cells of theanimal. The transgenic animal (including its progeny) will also have thetransgene fortuitously integrated into the chromosomes of somatic cells.

[0036] The TGF-β superfamily consists of multifunctional polypeptidesthat control proliferation, differentiation, and other functions in manycell types. Many of the peptides have regulatory, both positive andnegative, effects on other peptide growth factors. The structuralhomology between the GDF-11 protein of this invention and the members ofthe TGF-β family, indicates that GDF-11 is a new member of the family ofgrowth and differentiation factors. Based on the known activities ofmany of the other members, it can be expected that GDF-11 will alsopossess biological activities that will make it useful as a diagnosticand therapeutic reagent.

[0037] In particular, certain members of this superfamily haveexpression patterns or possess activities that relate to the function ofthe nervous system. For example, the inhibins and activins have beenshown to be expressed in the brain (Meunier, et al., Proc. Natl. Acad.Sci., USA, 85:247, 1988; Sawchenko, et al., Nature, 334:615, 1988), andactivin has been shown to be capable of functioning as a nerve cellsurvival molecule (Schubert, et al., Nature, 344:868, 1990). Anotherfamily member, namely, GDF-1, is nervous system-specific in itsexpression pattern (Lee, S. J., Proc. Natl. Acad. Sci., USA, 88:4250,1991), and certain other family members, such as Vgr-1 (Lyons, et al.,Proc. Natl. Acad. Sci., USA, 86:4554, 1989; Jones, et al., Development,111:531, 1991), OP-1 (Ozkaynak, et al., J. Biol. Chem., 267:25220,1992), and BMP-4 (Jones, et al., Development, 111:531, 1991), are alsoknown to be expressed in the nervous system. Because it is known thatskeletal muscle produces a factor or factors that promote the survivalof motor neurons (Brown, Trends Neurosci., 7:10, 1984), the expressionof GDF-11 in muscle suggests that one activity of GDF-11 may be as atrophic factor for neurons. In this regard, GDF-11 may have applicationsin the treatment of neurodegenerative diseases, such as amyotrophiclateral sclerosis or muscular dystrophy, or in maintaining cells ortissues in culture prior to transplantation.

[0038] The expression of GDF-11 in adipose tissue also raises thepossibility of applications for GDF-11 in the treatment of obesity or ofdisorders related to abnormal proliferation of adipocytes. In thisregard, TGF-β has been shown to be a potent inhibitor of adipocytedifferentiation in vitro (Ignotz and Massague, Proc. Natl. Acad. Sci.,USA 82:8530, 1985).

[0039] Polypeptides, Polynucleotides, Vectors and Host Cells

[0040] The invention provides substantially pure GDF-11 polypeptide andisolated polynucleotides that encode GDF-11. The term “substantiallypure” as used herein refers to GDF-11 which is substantially free ofother proteins, lipids, carbohydrates or other materials with which itis naturally associated. One skilled in the art can purify GDF-11 usingstandard techniques for protein purification. The substantially purepolypeptide will yield a single major band on a non-reducingpolyacrylamide gel. The purity of the GDF-11 polypeptide can also bedetermined by amino-terminal amino acid sequence analysis. GDF-11polypeptide includes functional fragments of the polypeptide, as long asthe activity of GDF-11 remains. Smaller peptides containing thebiological activity of GDF-11 are included in the invention.

[0041] The invention provides polynucleotides encoding the GDF-11protein. These polynucleotides include DNA, cDNA and RNA sequences whichencode GDF-11. It is understood that all polynucleotides encoding all ora portion of GDF-11 are also included herein, as long as they encode apolypeptide with GDF-11 activity. Such polynucleotides include naturallyoccurring, synthetic, and intentionally manipulated polynucleotides. Forexample, GDF-11 polynucleotide may be subjected to site-directedmutagenesis. The polynucleotide sequence for GDF11 also includesantisense sequences. The polynucleotides of the invention includesequences that are degenerate as a result of the genetic code. There are20 natural amino acids, most of which are specified by more than onecodon. Therefore, all degenerate nucleotide sequences are included inthe invention as long as the amino acid sequence of GDF-11 polypeptideencoded by the nucleotide sequence is functionally unchanged.

[0042] Specifically disclosed herein is a DNA sequence containing thehuman GDF-11 gene. The sequence contains an open reading frame encodinga polypeptide 407 amino acids in length. The sequence contains aputative RXXR proteolytic cleavage site at amino acids 295-298. Cleavageof the precursor at this site would generate an active C-terminalfragment 109 amino acids in length with a predicted molecular weight ofapproximately 12,500 kD. Also disclosed herein is a partial murinegenomic sequence. Preferably, the human GDF-11 nucleotide sequence isSEQ ID NO:1 and the mouse nucleotide sequence is SEQ ID NO:3.

[0043] The polynucleotide encoding GDF-11 includes SEQ ID NO:1 and 3, aswell as nucleic acid sequences complementary to SEQ ID NO's:1 and 3. Acomplementary sequence may include an antisense nucleotide. When thesequence is RNA, the deoxynucleotides A, G, C, and T of SEQ ID NO:1 and3 are replaced by ribonucleotides A, G, C, and U, respectively. Alsoincluded in the invention are fragments of the above-described nucleicacid sequences that are at least 15 bases in length, which is sufficientto permit the fragment to selectively hybridize to DNA that encodes theprotein of SEQ ID NO: 2 or 4 under physiological conditions (e.g., understringent conditions).

[0044] In nucleic acid hybridization reactions, the conditions used toachieve a particular level of stringency will vary, depending on thenature of the nucleic acids being hybridized. For example, the length,degree of complementarity, nucleotide sequence composition (e.g., GC v.AT content), and nucleic acid type (e.g., RNA v. DNA) of the hybridizingregions of the nucleic acids can be considered in selectinghybridization conditions. An additional consideration is whether one ofthe nucleic acids immobilized, for example, on a filter.

[0045] An example of progressively higher stringency conditions is asfollows: 2×SSC/0.1% SDS at about is room temperature (hybridizationconditions); 0.2×SSC/0.1% SDS at about room temperature (low stringencyconditions); 0.2×SSC/0.1% SDS at about 42° C. (moderate stringencyconditions); and 0.1×SSC at about 68° C. (high stringency conditions).Washing can be carried out using only one of these conditions, e.g.,high stringency conditions, or each of the conditions can be used, e.g.,for 10-15 minutes each, in the order listed above, repeating any or allof the steps listed. However, as mentioned above, optimal conditionswill vary, depending on the particular hybridization reaction involved,and can be determined empirically.

[0046] The C-terminal region of GDF-11 following the putativeproteolytic processing site shows significant homology to the knownmembers of the TGF-β superfamily. The GDF-11 sequence contains most ofthe residues that are highly conserved in other family members (see FIG.1). Like the TGF-βs and inhibin βs, GDF-11 contains an extra pair ofcysteine residues in addition to the 7 cysteines found in virtually allother family members. Among the known family members, GDF-11 is mosthomologous to GDF-8 (92% sequence identity) (see FIG. 3).

[0047] Minor modifications of the recombinant GDF-11 primary amino acidsequence may result in proteins which have substantially equivalentactivity as compared to the GDF-11 polypeptide described herein. Suchmodifications may be deliberate, as by site-directed mutagenesis, or maybe spontaneous. All of the polypeptides produced by these modificationsare included herein as long as the biological activity of GDF-11 stillexists. Further, deletion of one or more amino acids can also result ina modification of the structure of the resultant molecule withoutsignificantly altering its biological activity. This can lead to thedevelopment of a smaller active molecule which would have broaderutility. For example, one can remove amino or carboxy terminal aminoacids which are not required for GDF-11 biological activity.

[0048] The nucleotide sequence encoding the GDF-11 polypeptide of theinvention includes the disclosed sequence and conservative variationsthereof. The term “conservative variation” as used herein denotes thereplacement of an amino acid residue by another, biologically similarresidue. Examples of conservative variations include the substitution ofone hydrophobic residue such as isoleucine, valine, leucine ormethionine for another, or the substitution of one polar residue foranother, such as the substitution of arginine for lysine, glutamic foraspartic acid, or glutamine for asparagine, and the like. The term“conservative variation” also includes the use of a substituted aminoacid in place of an unsubstituted parent amino acid provided thatantibodies raised to the substituted polypeptide also immunoreact withthe unsubstituted polypeptide.

[0049] DNA sequences of the invention can be obtained by severalmethods. For example, the DNA can be isolated using hybridizationtechniques which are well known in the art. These include, but are notlimited to: 1) hybridization of genomic or cDNA libraries with probes todetect homologous nucleotide sequences, 2) polymerase chain reaction(PCR) on genomic DNA or cDNA using primers capable of annealing to theDNA sequence of interest, and 3) antibody screening of expressionlibraries to detect cloned DNA fragments with shared structuralfeatures.

[0050] Preferably the GDF-11 polynucleotide of the invention is derivedfrom a mammalian organism, and most preferably from mouse, rat, cow,pig, or human. GDF-11 polynucleotides from chicken, fish and otherspecies are also included herein. Screening procedures which rely onnucleic acid hybridization make it possible to isolate any gene sequencefrom any organism, provided the appropriate probe is available.Oligonucleotide probes, which correspond to a part of the sequenceencoding the protein in question, can be synthesized chemically. Thisrequires that short, oligopeptide stretches of amino acid sequence mustbe known. The DNA sequence encoding the protein can be deduced from thegenetic code, however, the degeneracy of the code must be taken intoaccount. It is possible to perform a mixed addition reaction when thesequence is degenerate. This includes a heterogeneous mixture ofdenatured double-stranded DNA. For such screening, hybridization ispreferably performed on either single-stranded DNA or denatureddouble-stranded DNA. Hybridization is particularly useful in thedetection of cDNA clones derived from sources where an extremely lowamount of mRNA sequences relating to the polypeptide of interest arepresent. In other words, by using stringent hybridization conditionsdirected to avoid non-specific binding, it is possible, for example, toallow the autoradiographic visualization of a specific cDNA clone by thehybridization of the target DNA to that single probe in the mixturewhich is its complete complement (Wallace, et al., Nucl. Acid Res.9:879, 1981).

[0051] The development of specific DNA sequences encoding GDF-11 canalso be obtained by: 1) isolation of double-stranded DNA sequences fromthe genomic DNA; 2) chemical manufacture of a DNA sequence to providethe necessary codons for the polypeptide of interest; and 3) in vitrosynthesis of a doublestranded DNA sequence by reverse transcription ofmRNA isolated from a eukaryotic donor cell. In the latter case, adouble-stranded DNA complement of mRNA is eventually formed which isgenerally referred to as cDNA.

[0052] Of the three above-noted methods for developing specific DNAsequences for use in recombinant procedures, the isolation of genomicDNA isolates is the least common. This is especially true when it isdesirable to obtain the microbial expression of mammalian polypeptidesdue to the presence of introns.

[0053] The synthesis of DNA sequences is frequently the method of choicewhen the entire sequence of amino acid residues of the desiredpolypeptide product is known. When the entire sequence of amino acidresidues of the desired polypeptide is not known, the direct synthesisof DNA sequences is not possible and the method of choice is thesynthesis of cDNA sequences. Among the standard procedures for isolatingcDNA sequences of interest is the formation of plasmid- orphage-carrying cDNA libraries which are derived from reversetranscription of mRNA which is abundant in donor cells that have a highlevel of genetic expression. When used in combination with polymerasechain reaction technology, even rare expression products can be cloned.In those cases where significant portions of the amino acid sequence ofthe polypeptide are known, the production of labeled single ordouble-stranded DNA or RNA probe sequences duplicating a sequenceputatively present in the target cDNA may be employed in DNA/DNAhybridization procedures which are carried out on cloned copies of thecDNA which have been denatured into a single-stranded form (Jay, et al.,Nucl. Acid Res., 11:2325, 1983).

[0054] A cDNA expression library, such as lambda gt11, can be screenedindirectly for GDF-11 peptides having at least one epitope, usingantibodies specific for GDF-11. Such antibodies can be eitherpolyclonally or monoclonally derived and used to detect expressionproduct indicative of the presence of GDF-11 cDNA.

[0055] DNA sequences encoding GDF-11 can be expressed in vitro by DNAtransfer into a suitable host cell. “Host cells” are cells in which avector can be propagated and its DNA expressed. The term also includesany progeny of the subject host cell. It is understood that all progenymay not be identical to the parental cell since there may be mutationsthat occur during replication. However, such progeny are included whenthe term “host cell” is used. Methods of stable transfer, meaning thatthe foreign DNA is continuously maintained in the host, are known in theart.

[0056] In the present invention, the GDF-11 polynucleotide sequences maybe inserted into a recombinant expression vector. The term “recombinantexpression vector” refers to a plasmid, virus or other vehicle known inthe art that has been manipulated by insertion or incorporation of theGDF-11 genetic sequences. Such expression vectors contain a promotersequence which facilitates the efficient transcription of the insertedgenetic sequence of the host. The expression vector typically containsan origin of replication, a promoter, as well as specific genes whichallow phenotypic selection of the transformed cells. Vectors suitablefor use in the present invention include, but are not limited to theT7-based expression vector for expression in bacteria (Rosenberg, etal., Gene, 56:125, 19117), the pMSXND expression vector for expressionin mammalian cells (Lee and Nathans, J. Biol. Chem., 263:3521, 1988) andbaculovirus-derived vectors for expression in insect cells. The DNAsegment can be present in the vector operably linked to regulatoryelements, for example, a promoter (e.g., T7, metallothionein 1, orpolyhedrin promoters).

[0057] Polynucleotide sequences encoding GDF-11 can be expressed ineither prokaryotes or eukaryotes. Hosts can include microbial, yeast,insect and mammalian organisms. Methods of expressing DNA sequenceshaving eukaryotic or viral sequences in prokaryotes are well known inthe art. Biologically functional viral and plasmid DNA vectors capableof expression and replication in a host are known in the art. Suchvectors are used to incorporate DNA sequences of the invention.Preferably, the mature C-terminal region of GDF-11 is expressed from acDNA clone containing the entire coding sequence of GDF-11.Alternatively, the C-terminal portion of GDF-11 can be expressed as afusion protein with the pro-region of another member of the TGF-β familyor co-expressed with another pro-region (see for example, Hammonds, etal., Molec. Endocrin., 5:149, 1991; Gray, A., and Mason, A., Science,247:1328, 1990).

[0058] Transformation of a host cell with recombinant DNA may be carriedout by conventional techniques as are well known to those skilled in theart. Where the host is prokaryotic, such as E. coli, competent cellswhich are capable of DNA uptake can be prepared from cells harvestedafter exponential growth phase and subsequently treated by the CaCl₂method using procedures well known in the art. Alternatively, MgCl₂ orRbCl can be used. Transformation can also be performed after forming aprotoplast of the host cell if desired.

[0059] When the host is a eukaryote, such methods of transfection of DNAas calcium phosphate co-precipitates, conventional mechanical proceduressuch as microinjection, electroporation, insertion of a plasmid encasedin liposomes, or virus vectors may be used. Eukaryotic cells can also becotransformed with DNA sequences encoding the GDF-11 of the invention,and a second foreign DNA molecule encoding a selectable phenotype, suchas the herpes simplex thymidine kinase gene. Another method is to use aeukaryotic viral vector, such as simian virus 40 (SV40) or bovinepapilloma virus, to transiently infect or transform eukaryotic cells andexpress the protein. (see for example, Eukaryotic Viral Vectors, ColdSpring Harbor Laboratory, Gluzman ed., 1982).

[0060] Isolation and purification of microbial expressed polypeptide, orfragments thereof, provided by the invention, may be carried out byconventional means including preparative chromatography andimmunological separations involving monoclonal or polyclonal antibodies.

[0061] GDF-11 Antibodies and Methods of Use

[0062] The invention includes antibodies immunoreactive with GDF-11polypeptide or functional fragments thereof. Antibody which consistsessentially of pooled monoclonal antibodies with different epitopicspecificities, as well as distinct monoclonal antibody preparations areprovided. Monoclonal antibodies are made from antigen containingfragments of the protein by methods well known to those skilled in theart (Kohler, et al., Nature, 256:495, 1975). The term antibody as usedin this invention is meant to include intact molecules as well asfragments thereof, such as Fab and F(ab′)₂, Fv and SCA fragments whichare capable of binding an epitopic determinant on GDF-11.

[0063] (1) An Fab fragment consists of a monovalent antigen-bindingfragment of an antibody molecule, and can be produced by digestion of awhole antibody molecule with the enzyme papain, to yield a fragmentconsisting of an intact light chain and a portion of a heavy chain.

[0064] (2) An Fab′ fragment of an antibody molecule can be obtained bytreating a whole antibody molecule with pepsin, followed by reduction,to yield a molecule consisting of an intact light chain and a portion ofa heavy chain. Two Fab′ fragments are obtained per antibody moleculetreated in this manner.

[0065] (3) An (Fab′)₂ fragment of an antibody can be obtained bytreating a whole antibody molecule with the enzyme pepsin, withoutsubsequent reduction. A (Fab′)₂ fragment is a dimer of two Fab′fragments, held together by two disulfide bonds.

[0066] (4) An Fv fragment is defined as a genetically engineeredfragment containing the variable region of a light chain and thevariable region of a heavy chain expressed as two chains.

[0067] (5) A single chain antibody (“SCA”) is a genetically engineeredsingle chain molecule containing the variable region of a light chainand the variable region of a heavy chain, linked by a suitable, flexiblepolypeptide linker.

[0068] As used in this invention, the term “epitope” refers to anantigenic determinant on an antigen, such as a GDF-11 polypeptide, towhich the paratope of an antibody, such as an GDF-11-specific antibody,binds. Antigenic determinants usually consist of chemically activesurface groupings of molecules, such as amino acids or sugar sidechains, and can have specific three-dimensional structuralcharacteristics, as well as specific charge characteristics.

[0069] As is mentioned above, antigens that can be used in producingGDF-11-specific antibodies include GDF-11 polypeptides or GDF-11polypeptide fragments. The polypeptide or peptide used to immunize ananimal can be obtained by standard recombinant, chemical synthetic, orpurification methods. As is well known in the art, in order to increaseimmunogenicity, an antigen can be conjugated to a carrier protein.Commonly used carriers include keyhole limpet hemocyanin (KLH),thyroglobulin, bovine serum albumin (BSA), and tetanus toxoid. Thecoupled peptide is then used to immunize the animal (e.g., a mouse, arat, or a rabbit). In addition to such carriers, well known adjuvantscan be administered with the antigen to facilitate induction of a strongimmune response.

[0070] The term “cell-proliferative disorder” denotes malignant as wellas non-malignant cell populations which often appear to differ from thesurrounding tissue both morphologically and genotypically. Malignantcells (i.e. cancer) develop as a result of a multistep process. TheGDF-11 polynucleotide that is an antisense molecule or that encodes adominant negative GDF-11 is useful in treating malignancies of thevarious organ systems, particularly, for example, cells in muscle, bone,kidney or adipose tissue. Essentially, any disorder which isetiologically linked to altered expression of GDF-11 could be consideredsusceptible to treatment with a GDF-11 agent (e.g., a suppressing orenhancing agent). One such disorder is a malignant cell proliferativedisorder, for example.

[0071] The invention provides a method for detecting a cellproliferative disorder of muscle, bone, kidney, uterine or neuraltissue, for example, which comprises contacting an anti-GDF-11 antibodywith a cell suspected of having a GDF-11 associated disorder anddetecting binding to the antibody. The antibody reactive with GDF-11 islabeled with a compound which allows detection of binding to GDF-11. Forpurposes of the invention, an antibody specific for GDF-11 polypeptidemay be used to detect the level of GDF-11 in biological fluids andtissues. Any specimen containing a detectable amount of antigen can beused. A preferred sample of this invention is muscle, bone, kidney,uterus, spleen, thymus, or neural tissue. The level of GDF-11 in thesuspect cell can be compared with the level in a normal cell todetermine whether the subject has a GDF-11-associated cell proliferativedisorder. Such methods of detection are also useful using nucleic acidhybridization to detect the level of GDF-11 mRNA in a sample or todetect an altered GDF-11 gene. Preferably the subject is human.

[0072] The antibodies of the invention can be used in any subject inwhich it is desirable to administer in vitro or in vivo immunodiagnosisor immunotherapy. The antibodies of the invention are suited for use,for example, in immunoassays in which they can be utilized in liquidphase or bound to a solid phase carrier. In addition, the antibodies inthese immunoassays can be detectably labeled in various ways. Examplesof types of immunoassays which can utilize antibodies of the inventionare competitive and non-competitive immunoassays in either a direct orindirect format. Examples of such immunoassays are the radioimmunoassay(RIA) and the sandwich (immunometric) assay. Detection of the antigensusing the antibodies of the invention can be done utilizing immunoassayswhich are run in either the forward, reverse, or simultaneous modes,including immunohistochemical assays on physiological samples. Those ofskill in the art will know, or can readily discern, other immunoassayformats without undue experimentation.

[0073] The antibodies of the invention can be bound to many differentcarriers and used to detect the presence of an antigen comprising thepolypeptide of the invention. Examples of well-known carriers includeglass, polystyrene, polypropylene, polyethylene, dextran, nylon,amylases, natural and modified celluloses, polyacrylamides, agaroses andmagnetite. The nature of the carrier can be either soluble or insolublefor purposes of the invention. Those skilled in the art will know ofother suitable carriers for binding antibodies, or will be able toascertain such, using routine experimentation.

[0074] There are many different labels and methods of labeling known tothose of ordinary skill in the art. Examples of the types of labelswhich can be used in the present invention include enzymes,radioisotopes, fluorescent compounds, colloidal metals, chemiluminescentcompounds, phosphorescent compounds, and bioluminescent compounds. Thoseof ordinary skill in the art will know of other suitable labels forbinding to the antibody, or will be able to ascertain such, usingroutine experimentation.

[0075] Another technique which may also result in greater sensitivityconsists of coupling the antibodies to low molecular weight haptens.These haptens can then be specifically detected by means of a secondreaction. For example, it is common to use such haptens as biotin, whichreacts with avidin, or dinitrophenyi, puridoxal, and fluorescein, whichcan react with specific antihapten antibodies.

[0076] In using the monoclonal antibodies of the invention for the invivo detection of antigen, the detectably labeled antibody is given adose which is diagnostically effective. The term “diagnosticallyeffective” means that the amount of detectably labeled monoclonalantibody is administered in sufficient quantity to enable detection ofthe site having the antigen comprising a polypeptide of the inventionfor which the monoclonal antibodies are specific.

[0077] The concentration of detectably labeled monoclonal antibody whichis administered should be sufficient such that the binding to thosecells having the polypeptide is detectable compared to the background.Further, it is desirable that the detectably labeled monoclonal antibodybe rapidly cleared from the circulatory system in order to give the besttarget-to-background signal ratio.

[0078] As a rule, the dosage of detectably labeled monoclonal antibodyfor in vivo diagnosis will vary depending on such factors as age, sex,and extent of disease of the individual. Such dosages may vary, forexample, depending on whether multiple injections are given, antigenicburden, and other factors known to those of skill in the art.

[0079] For in vivo diagnostic imaging, the type of detection instrumentavailable is a major factor in selecting a given radioisotope. Theradioisotope chosen must have a type of decay which is detectable for agiven type of instrument. Still another important factor in selecting aradioisotope for in vivo diagnosis is that deleterious radiation withrespect to the host is minimized. Ideally, a radioisotope used for invivo imaging will lack a particle emission, but produce a large numberof photons in the 140-250 keV range, which may readily be detected byconventional gamma cameras.

[0080] For in vivo diagnosis radioisotopes may be bound toimmunoglobulin either directly or indirectly by using an intermediatefunctional group. intermediate functional groups which often are used tobind radioisotopes which exist as metallic ions to immunoglobulins arethe bifunctional chelating agents such as diethylenetriaminepentaceticacid (DTPA) and ethylenediaminetetraacetic acid (EDTA) and similarmolecules. Typical examples of metallic ions which can be bound to themonoclonal antibodies of the invention are ¹¹¹In, ⁹⁷Ru, ⁶⁷Ga, ⁶⁸Ga,⁷²As, ⁸⁹Zr and ²⁰¹Tl.

[0081] The monoclonal antibodies of the invention can also be labeledwith a paramagnetic isotope for purposes of in vivo diagnosis, as inmagnetic resonance imaging (MRI) or electron spin resonance (ESR). Ingeneral, any conventional method for visualizing diagnostic imaging canbe utilized. Usually gamma and positron emitting radioisotopes are usedfor camera imaging and paramagnetic isotopes for MRI. Elements which areparticularly useful in such techniques include ¹⁵⁷Gd, ⁵⁵Mn, ¹⁶²Dy, ⁵²Cr,and ⁵⁶Fe.

[0082] The monoclonal antibodies of the invention can be used in vitroand in vivo to monitor the course of amelioration of a GDF-11-associateddisease in a subject. Thus, for example, by measuring the increase ordecrease in the number of cells expressing antigen comprising apolypeptide of the invention or changes in the concentration of suchantigen present in various body fluids, it would be possible todetermine whether a particular therapeutic regimen aimed at amelioratingthe GDF-11-associated disease is effective. The term “ameliorate”denotes a lessening of the detrimental effect of the GDF-11-associateddisease in the subject receiving therapy.

[0083] Additional Methods of Treatment and Diagnosis

[0084] The present invention identifies a nucleotide sequence that canbe expressed in an altered manner as compared to expression in a normalcell, therefore it is possible to design appropriate therapeutic ordiagnostic techniques directed to this sequence. Treatment includesadministration of a reagent which modulates activity. The term“modulate” envisions the suppression or expression of GDF-11 when it isover-expressed, or augmentation of GDF-11 expression when it isunderexpressed. When a muscle-associated disorder is associated withGDF-11 overexpression, such suppressive reagents as antisense GDF-11polynucleotide sequence, dominant negative sequences or GDF-11 bindingantibody can be introduced into a cell. In addition, an anti-idiotypeantibody which binds to a monoclonal antibody which binds GDF-11 of theinvention, or an epitope thereof, may also be used in the therapeuticmethod of the invention. Alternatively, when a cell proliferativedisorder is associated with underexpression or expression of a mutantGDF-11 polypeptide, a sense polynucleotide sequence (the DNA codingstrand) or GDF-11 polypeptide can be introduced into the cell. Suchmuscle-associated disorders include cancer, muscular dystrophy, spinalcord injury, traumatic injury, congestive obstructive pulmonary disease(COPD), AIDS or cachecia. Neurodegenerative, musculoskeletal, and kidneydisorders are also envisioned as treated by the method of the invention.In addition, the method of the invention can be used in the treatment ofobesity or of disorders related to abnormal proliferation of adipocytes.One of skill in the art can determine whether or not a particulartherapeutic course of treatment is successful by several methodsdescribed herein (e.g., muscle fiber analysis or biopsy; determinationof fat content). The present examples demonstrate that the methods ofthe invention are useful for decreasing fat content, and therefore wouldbe useful in the treatment of obesity and related disorders (e.g.,diabetes).

[0085] Thus, where a cell-proliferative disorder is associated with theexpression of GDF-11, nucleic acid sequences that interfere with GDF-11expression at the translational level can be used. This approachutilizes, for example, antisense nucleic acid and ribozymes to blocktranslation of a specific GDF-11 mRNA, either by masking that mRNA withan antisense nucleic acid or by cleaving it with a ribozyme. Suchdisorders include neurodegenerative diseases, for example. In addition,dominant-negative GDF-11 mutants would be useful to actively interferewith function of “normal” GDF-11.

[0086] Antisense nucleic acids are DNA or RNA molecules that arecomplementary to at least a portion of a specific mRNA molecule(Weintraub, Scientific American, 262:40, 1990). In the cell, theantisense nucleic acids hybridize to the corresponding mRNA, forming adouble-stranded molecule. The antisense nucleic acids interfere with thetranslation of the mRNA, since the cell will not translate a mRNA thatis double-stranded.

[0087] Antisense oligomers of about 15 nucleotides are preferred, sincethey are easily synthesized and are less likely to cause problems thanlarger molecules when introduced into the target GDF-11-producing cell.The use of antisense methods to inhibit the in vitro translation ofgenes is well known in the art (Marcus-Sakura, Anal. Biochem., 172:289,1988).

[0088] Ribozymes are RNA molecules possessing the ability tospecifically cleave other single-stranded RNA in a manner analogous toDNA restriction endonucleases. Through the modification of nucleotidesequences which encode these RNAs, it is possible to engineer moleculesthat recognize specific nucleotide sequences in an RNA molecule andcleave it (Cech, J. Amer. Med. Assn., 260:3030, 1988). A major advantageof this approach is that, because they are sequence-specific, only mRNAswith particular sequences are inactivated.

[0089] There are two basic types of ribozymes namely, tetrahymena-type(Hasselhoff, Nature, 334:585, 1988) and “hammerhead”-type.Tetrahymena-type ribozymes recognize sequences which are four bases inlength, while “hammerhead”-type ribozymes recognize base sequences 11-18bases in length. The longer the recognition sequence, the greater thelikelihood that the sequence will occur exclusively in the target mRNAspecies. Consequently, hammerhead-type ribozymes are preferable totetrahymena-type ribozymes for inactivating a specific mRNA species and18-based recognition sequences are preferable to shorter recognitionsequences.

[0090] In another embodiment of the present invention, a nucleotidesequence encoding a GDF-11 dominant negative protein is provided. Forexample, a genetic construct that contain such a dominant negativeencoding gene may be operably linked to a promoter, such as atissue-specific promoter. For example, a skeletal muscle specificpromoter (e.g., human skeletal muscle α-actin promoter) ordevelopmentally specific promoter (e.g., MyHC 3, which is restricted inskeletal muscle to the embryonic period of development, or an induciblepromoter (e.g., the orphan nuclear receptor TIS1).

[0091] Such constructs are useful in methods of modulating a subject'sskeletal mass. For example, a method include transforming an organism,tissue, organ or cell with a genetic construct encoding a dominantnegative GDF-11 protein and suitable promoter in operable linkage andexpressing the dominant negative encoding GDF-11 gene, therebymodulating muscle mass, bone content and/or kidney growth by interferingwith wild-type GDF-11 activity.

[0092] GDF-11 most likely forms dimers, homodimers or heterodimers andmay even form heterodimers with other GDF family members, such as GDF-11(see Example 4). Hence, while not wanting to be bound by a particulartheory, the dominant negative effect described herein may involve theformation of non-functional homodimers or heterodimers of dominantnegative and wild-type GDF-11 monomers. More specifically, it ispossible that any non-functional homodimer or any heterodimer formed bythe dimerization of wild-type and dominant negative GDF-11 monomersproduces a dominant effect by: 1) being synthesized but not processed orsecreted; 2) inhibiting the secretion of wild type GDF-11; 3) preventingnormal proteolytic cleavage of the preprotein thereby producing anonfumctional GDF-11 molecule; 4) altering the affinity of thenon-functional dimer (e.g., homodimeric or heterodimeric GDF-11) to areceptor or generating an antagonistic form of GDF-11 that binds areceptor without activating it; or 5) inhibiting the intracellularprocessing or secretion of GDF-11 related or TGF-β family proteins.

[0093] Non-functional GDF-11 can function to inhibit the growthregulating actions of GDF-11 on muscle cells that include a dominantnegative GDF-11 gene. Deletion or missense dominant negative forms ofGDF-11 that retain the ability to form dimers with wild-type GDF-11protein but do not function as wild-type GDF-11 proteins may be used toinhibit the biological activity of endogenous wild-type GDF-11. Forexample, in one embodiment, the proteolytic processing site of GDF-11may be altered (e.g., deleted) resulting in a GDF-11 molecule able toundergo subsequent dimerization with endogenous wild-type GDF-11 butunable to undergo further processing into a mature GDF-11 form.Alternatively, a non-functional GDF-11 can function as a monomericspecies to inhibit the growth regulating actions of GDF-11 on musclecells, bone cells and kidney cells at any point in a tissue's ororganism's development.

[0094] Any genetic recombinant method in the art may be used, forexample, recombinant viruses may be engineered to express a dominantnegative form of GDF-11 which may be used to inhibit the activity ofwild-type GDF-11. Such viruses may be used therapeutically for treatmentof diseases resulting from aberrant over-expression or activity ofGDF-11 protein, such as in denervation hypertrophy or as a means ofcontrolling GDF-11 expression when treating disease conditions involvingmuscle, such as in musculodegenerative diseases or in tissue repair dueto trauma or in modulating GDF-11 expression in animal husbandry (e.g.,transgenic animals for agricultural purposes).

[0095] In addition, the expression of GDF-11 may be used, for example tohelp in kidney development. The method includes administering atherapeutically effective amount of a GDF-11 agent to the subject,thereby promoting kidney cell growth and differentiation in kidneytissue. The agent may be an antagonist or agonist of GDF-11 activity.For example, the agent may include a GDF-11 antisense molecule or adominant negative polypeptide.

[0096] The invention provides a method for treating a muscle, kidney(chronic or acute) or adipose tissue disorder in a subject. The methodincludes administering a therapeutically effective amount of a GDF-11agent to the subject, thereby inhibiting abnormal growth of muscle oradipose tissue or stimulating growth in kidney tissue. The GDF-11 agentmay include a GDF-11 antisense molecule or a dominant negativepolypeptide, for example. A “therapeutically effective amount” of aGDF-11 agent is that amount that ameliorates symptoms of the disorder orinhibits GDF-11 induced growth of muscle, for example, as compared witha normal subject.

[0097] The present invention also provides gene therapy for thetreatment of cell proliferative or immunologic disorders which aremediated by GDF-11 protein. Such therapy would achieve its therapeuticeffect by introduction of the GDF-11 antisense polynucleotide ordominant negative encoding polynucleotide sequences into cells havingthe proliferative disorder. Delivery of antisense GDF-11 polynucleotidecan be achieved using a recombinant expression vector such as a chimericvirus or a colloidal dispersion system. Especially preferred fortherapeutic delivery of antisense or dominant negative sequences is theuse of targeted liposomes. In contrast, when it is desirable to enhanceGDF-11 production, a “sense” GDF-11 polynucleotide is introduced intothe appropriate cell(s).

[0098] Various viral vectors which can be utilized for gene therapy astaught herein include adenovirus, herpes virus, vaccinia, or,preferably, an RNA virus such as a retrovirus. Preferably, theretroviral vector is a derivative of a murine or avian retrovirus.Examples of retroviral vectors in which a single foreign gene can beinserted include, but are not limited to: Moloney murine leukemia virus(MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumorvirus (MuMTV), and Rous Sarcoma Virus (RSV). A number of additionalretroviral vectors can incorporate multiple genes. All of these vectorscan transfer or incorporate a gene for a selectable marker so thattransduced cells can be identified and generated. By inserting a GDF-11sequence of interest into the viral vector, along with another genewhich encodes the ligand for a receptor on a specific target cell, forexample, the vector is now target specific. Retroviral vectors can bemade target specific by attaching, for example, a sugar, a glycolipid,or a protein. Preferred targeting is accomplished by using an antibodyto target the retroviral vector. Those of skill in the art will know of,or can readily ascertain without undue experimentation, specificpolynucleotide sequences which can be inserted into the retroviralgenome or attached to a viral envelope to allow target specific deliveryof the retroviral vector containing the GDF-11 antisense polynucleotide.

[0099] Since recombinant retroviruses are defective, they requireassistance in order to produce infectious vector particles. Thisassistance can be provided, for example, by using helper cell lines thatcontain plasmids encoding all of the structural genes of the retrovirusunder the control of regulatory sequences within the LTR. These plasmidsare missing a nucleotide sequence which enables the packaging mechanismto recognize an RNA transcript for encapsulation. Helper cell lineswhich have deletions of the packaging signal include, but are notlimited to ψ2, PA317 and PA12, for example. These cell lines produceempty virions, since no genome is packaged. If a retroviral vector isintroduced into such cells in which the packaging signal is intact, butthe structural genes are replaced by other genes of interest, the vectorcan be packaged and vector virion produced.

[0100] Alternatively, NIH 3T3 or other tissue culture cells can bedirectly transfected with plasmids encoding the retroviral structuralgenes gag, pol and env, by conventional calcium phosphate transfection.These cells are then transfected with the vector plasmid containing thegenes of interest. The resulting cells release the retroviral vectorinto the culture medium.

[0101] Another targeted delivery system for GDF-11 antisensepolynucleotides is a colloidal dispersion system. Colloidal dispersionsystems include macromolecule complexes, nanocapsules, microspheres,beads, and lipid-based systems including oil-in-water emulsions,micelles, mixed micelles, and liposomes. The preferred colloidal systemof this invention is a liposome. Liposomes are artificial membranevesicles which are useful as delivery vehicles in vitro and in vivo. Ithas been shown that large unilamellar vesicles (LUV), which range insize from 0.2-4.0 μm can encapsulate a substantial percentage of anaqueous buffer containing large macromolecules. RNA, DNA and intactvirions can be encapsulated within the aqueous interior and be deliveredto cells in a biologically active form (Fraley, et al., Trends Biochem.Sci, 6:77, 19111). In addition to mammalian cells, liposomes have beenused for delivery of polynucleotides in plant, yeast and bacterialcells. in order for a liposome to be an efficient gene transfer vehicle,the following characteristics should be present: (1) encapsulation ofthe genes of interest at high efficiency while not compromising theirbiological activity; (2) preferential and substantial binding to atarget cell in comparison to non-target cells; (3) delivery of theaqueous contents of the vesicle to the target cell cytoplasm at highefficiency; and (4) accurate and effective expression of geneticinformation (Manning, et al., Biotechniques, 6:682, 1988).

[0102] The composition of the liposome is usually a combination ofphospholipids, particularly high-phase-transition-temperaturephospholipids, usually in combination with steroids, especiallycholesterol. Other phospholipids or other lipids may also be used. Thephysical characteristics of liposomes depend on pH, ionic strength, andthe presence of divalent cations.

[0103] Examples of lipids useful in liposome production includephosphatidyl compounds, such as phosphatidyiglycerol,phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine,sphingolipids, cerebrosides, and gangliosides. Particularly useful arediacylphosphatidylglycerols, where the lipid moiety contains from 14-18carbon atoms, particularly from 16-18 carbon atoms, and is saturated.Illustrative phospholipids include egg phosphatidylchloline,dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.

[0104] The targeting of liposomes can be classified based on anatomicaland mechanistic factors. Anatomical classification is based on the levelof selectivity, for example, organ-specific, cell-specific, andorganelle-specific. Mechanistic targeting can be distinguished basedupon whether it is passive or active. Passive targeting utilizes thenatural tendency of liposomes to distribute to cells of thereticulo-endothelial system (RES) in organs which contain sinusoidalcapillaries. Active targeting, on the other hand, involves alteration ofthe liposome by coupling the liposome to a specific ligand such as amonoclonal antibody, sugar, glycolipid, or protein, or by changing thecomposition or size of the liposome in order to achieve targeting toorgans and cell types other than the naturally occurring sites oflocalization.

[0105] The surface of the targeted delivery system may be modified in avariety of ways. In the case of a liposomal targeted delivery system,lipid groups can be incorporated into the lipid bilayer of the liposomein order to maintain the targeting ligand in stable association with theliposomal bilayer. Various linking groups can be used for joining thelipid chains to the targeting ligand.

[0106] Due to the expression of GDF-11 in muscle and adipose tissue,there are a variety of applications using the polypeptide,polynucleotide, and antibodies of the invention, related to thesetissues. Such applications include treatment of cell proliferativedisorders involving these and other tissues, such as neural tissue. Inaddition, GDF-11 may be useful in various gene therapy procedures. Inembodiments where GDF-11 polypeptide is administered to a subject, thedosage range is about 0.1 ug/kg to 100 mg/kg; more preferably from about1 ug/kg to 75 mg/kg and most preferably from about 10 mg/kg to 50 mg/kg.

[0107] Chromosomal Location of GDF-11

[0108] The data in Example 6 shows that the human GDF-11 gene is locatedon chromosome 2. By comparing the chromosomal location of GDF-11 withthe map positions of various human disorders, it should be possible todetermine whether mutations in the GDF-11 gene are involved in theetiology of human diseases. For example, an autosomal recessive form ofjuvenile amyotrophic lateral sclerosis has been shown to map tochromosome 2 (Hentati, et al., Neurology, 42 [Suppl.3]:201, 1992). Moreprecise mapping of GDF-11 and analysis of DNA from these patients mayindicate that GDF-11 is, in fact, the gene affected in this disease. Inaddition, GDF-11 is useful for distinguishing chromosome 2 from otherchromosomes.

[0109] Transgenic Animals and Methods of Making the Same

[0110] Various methods to make the transgenic animals of the subjectinvention can be employed. Generally speaking, three such methods may beemployed. In one such method, an embryo at the pronuclear stage (a “onecell embryo”) is harvested from a female and the transgene ismicroinjected into the embryo, in which case the transgene will bechromosomally integrated into both the germ cells and somatic cells ofthe resulting mature animal. In another such method, embryonic stemcells are isolated and the transgene incorporated therein byelectroporation, plasmid transfection or microinjection, followed byreintroduction of the stem cells into the embryo where they colonize andcontribute to the germ line. Methods for microinjection of mammalianspecies is described in U.S. Pat. No. 4,873,191. In yet another suchmethod, embryonic cells are infected with a retrovirus containing thetransgene whereby the germ cells of the embryo have the transgenechromosomally integrated therein. When the animals to be made transgenicare avian, because avian fertilized ova generally go through celldivision for the first twenty hours in the oviduct, microinjection intothe pronucleus of the fertilized egg is problematic due to theinaccessibility of the pronucleus. Therefore, of the methods to maketransgenic animals described generally above, retrovirus infection ispreferred for avian species, for example as described in U.S. Pat. No.5,162,215. If microinjection is to be used with avian species, however,a recently published procedure by Love et al., (Biotechnology, Jan. 12,1994) can be utilized whereby the embryo is obtained from a sacrificedhen approximately two and one-half hours after the laying of theprevious laid egg, the transgene is microinjected into the cytoplasm ofthe germinal disc and the embryo is cultured in a host shell untilmaturity. When the animals to be made transgenic are bovine or porcine,microinjection can be hampered by the opacity of the ova thereby makingthe nuclei difficult to identify by traditional differentialinterference-contrast microscopy. To overcome this problem, the ova canfirst be centrifuged to segregate the pronuclei for bettervisualization.

[0111] The “non-human animals” of the invention bovine, porcine, ovineand avian animals (e.g., cow, pig, sheep, chicken). The “transgenicnon-human animals” of the invention are produced by introducing“transgenes” into the germline of the non-human animal. Embryonal targetcells at various developmental stages can be used to introducetransgenes. Different methods are used depending on the stage ofdevelopment of the embryonal target cell. The zygote is the best targetfor micro-injection. The use of zygotes as a target for gene transferhas a major advantage in that in most cases the injected DNA will beincorporated into the host gene before the first cleavage (Brinster etal., Proc. Natl. Acad. Sci. USA 82:4438-4442, 1985). As a consequence,all cells of the transgenic non-human animal will carry the incorporatedtransgene. This will in general also be reflected in the efficienttransmission of the transgene to offspring of the founder since 50% ofthe germ cells will harbor the transgene.

[0112] The term “transgenic” is used to describe an animal whichincludes exogenous genetic material within all of its cells. A“transgenic” animal can be produced by cross-breeding two chimericanimals which include exogenous genetic material within cells used inreproduction. Twenty-five percent of the resulting offspring will betransgenic i.e., animals which include the exogenous genetic materialwithin all of their cells in both alleles. 50% of the resulting animalswill include the exogenous genetic material within one allele and 25%will include no exogenous genetic material.

[0113] In the microinjection method useful in the practice of thesubject invention, the transgene is digested and purified free from anyvector DNA e.g. by gel electrophoresis. It is preferred that thetransgene include an operatively associated promoter which interactswith cellular proteins involved in transcription, ultimately resultingin constitutive expression. Promoters useful in this regard includethose from cytomegalovirus (CMV), Moloney leukemia virus (MLV), andherpes virus, as well as those from the genes encoding metallothionin,skeletal actin, P-enolpyruvate carboxylase (PEPCK), phosphoglycerate(PGK), DHFR, and thymidine kinase. Promoters for viral long terminalrepeats (LTRs) such as Rous Sarcoma Virus can also be employed. When theanimals to be made transgenic are avian, preferred promoters includethose for the chicken β-globin gene, chicken lysozyme gene, and avianleukosis virus. Constructs useful in plasmid transfection of embryonicstem cells will employ additional regulatory elements well known in theart such as enhancer elements to stimulate transcription, spliceacceptors, termination and polyadenylation signals, and ribosome bindingsites to permit translation.

[0114] Retroviral infection can also be used to introduce transgene intoa non-human animal, as described above. The developing non-human embryocan be cultured in vitro to the blastocyst stage. During this time, theblastomeres can be targets for retro viral infection (Jaenich, R., Proc.Natl. Acad. Sci USA 73:1260-1264, 1976). Efficient infection of theblastomeres is obtained by enzymatic treatment to remove the zonapellucida (Hogan, et al. (1986) in Manipulating the Mouse Embryo, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). The viralvector system used to introduce the transgene is typically areplication-defective retro virus carrying the transgene (Jahner, etal., Proc. Natl. Acad. Sci USA 82:6927-6931, 1985; Van der Putten, etal., Proc. Natl. Acad. Sci USA 82:6148-6152, 1985). Transfection iseasily and efficiently obtained by culturing the blastomeres on amonolayer of virus-producing cells (Van der Putten, supra; Stewart, etal., EMBO J. 6:383-388, 1987). Alternatively, infection can be performedat a later stage. Virus or virus-producing cells can be injected intothe blastocoele (D. Jahner et al., Nature 298:623-628, 1982). Most ofthe founders will be mosaic for the transgene since incorporation occursonly in a subset of the cells which formed the transgenic nonhumananimal. Further, the founder may contain various retro viral insertionsof the transgene at different positions in the genome which generallywill segregate in the offspring. In addition, it is also possible tointroduce transgenes into the germ line, albeit with low efficiency, byintrauterine retroviral infection of the midgestation embryo (D. Jahneret al., supra).

[0115] A third type of target cell for transgene introduction is theembryonal stem cell (ES). ES cells are obtained from pre-implantationembryos cultured in vitro and fused with embryos (M. J. Evans et al.Nature 292:154-156, 1981; M. O. Bradley et al., Nature 309: 255-258,1984; Gossler, et al., Proc. Natl. Acad. Sci USA 83: 9065-9069, 1986;and Robertson et al., Nature 322:445-448, 1986). Transgenes can beefficiently introduced into the ES cells by DNA transfection or by retrovirus-mediated transduction. Such transformed ES cells can thereafter becombined with blastocysts from a nonhuman animal. The ES cellsthereafter colonize the embryo and contribute to the germ line of theresulting chimeric animal. (For review see Jaenisch, R., Science 240:1468-1474, 1988).

[0116] “Transformed” means a cell into which (or into an ancestor ofwhich) has been introduced, by means of recombinant nucleic acidtechniques, a heterologous nucleic acid molecule. “Heterologous” refersto a nucleic acid sequence that either originates from another speciesor is modified from either its original form or the form primarilyexpressed in the cell.

[0117] “Transgene” means any piece of DNA which is inserted by artificeinto a cell, and becomes part of the genome of the organism (i.e.,either stably integrated or as a stable extrachromosomal element) whichdevelops from that cell. Such a transgene may include a gene which ispartly or entirely heterologous (i.e., foreign) to the transgenicorganism, or may represent a gene homologous to an endogenous gene ofthe organism. Included within this definition is a transgene created bythe providing of an RNA sequence which is transcribed into DNA and thenincorporated into the genome. The transgenes of the invention includeDNA sequences which encode GDF-11, and include GDF-sense and antisensepolynucleotides and dominant negative encoding polynculeotides, whichmay be expressed in a transgenic non-human animal. The term “transgenic”as used herein additionally includes any organism whose genome has beenaltered by in vitro manipulation of the early embryo or fertilized eggor by any transgenic technology to induce a specific gene knockout. Theterm “gene knockout” as used herein, refers to the targeted disruptionof a gene in vivo with complete loss of function that has been achievedby any transgenic technology familiar to those in the art. In oneembodiment, transgenic animals having gene knockouts are those in whichthe target gene has been rendered nonfunctional by an insertion targetedto the gene to be rendered non-functional by homologous recombination.As used herein, the term “transgenic” includes any transgenic technologyfamiliar to those in the art which can produce an organism carrying anintroduced transgene or one in which an endogenous gene has beenrendered non-functional or “knocked out.” An example of a transgene usedto “knockout” GDF-11 function in the present Examples is described inExample 6 and FIG. 9. Thus, in another embodiment, the inventionprovides a transgene wherein the entire mature C-terminal region ofGDF-11 is deleted.

[0118] The transgene to be used in the practice of the subject inventionis a DNA sequence comprising a modified GDF-11 coding sequence. In apreferred embodiment, the GDF-11 gene is disrupted by homologoustargeting in embryonic stem cells. For example, the entire matureC-terminal region of the GDF-11 gene may be deleted as described in theexamples below. Optionally, the GDF-11 disruption or deletion may beaccompanied by insertion of or replacement with other DNA sequences,such as a non-functional GDF-11 sequence. In other embodiments, thetransgene comprises DNA antisense to the coding sequence for GDF-11. Inanother embodiment, the transgene comprises DNA encoding an antibody orreceptor peptide sequence which is able to bind to GDF-11. The DNA andpeptide sequences of GDF-11 are known in the art, the sequences,localization and activity disclosed in WO95/08543 and pending U.S.patent application Ser. No. 08/706,958, filed on Sep. 3, 1996,incorporated by reference in its entirety. The disclosure of both ofthese applications are hereby incorporated herein by reference. Whereappropriate, DNA sequences that encode proteins having GDF-11 activitybut differ in nucleic acid sequence due to the degeneracy of the geneticcode may also be used herein, as may truncated forms, allelic variantsand interspecies homologues.

[0119] Therefore the invention also includes animals having heterozygousmutations in GDF-11. A heterozygote would likely have an intermediateincrease in muscle mass as compared to the homozygote.

[0120] After an embryo has been microinjected, colonized withtransfected embryonic stem cells or infected with a retroviruscontaining the transgene (except for practice of the subject inventionin avian species which is addressed elsewhere herein) the embryo isimplanted into the oviduct of a pseudopregnant female. The consequentprogeny are tested for incorporation of the transgene by Southern blotanalysis of blood samples using transgene specific probes. PCR isparticularly useful in this regard. Positive progeny (G0) are crossbredto produce offspring (G1) which are analyzed for transgene expression byNorthern blot analysis of tissue samples. To be able to distinguishexpression of like-species transgenes from expression of the animalsendogenous GDF-11 gene(s), a marker gene fragment can be included in theconstruct in the 3′ untranslated region of the transgene and theNorthern probe designed to probe for the marker gene fragment. The serumlevels of GDF-11 can also be measured in the transgenic animal toestablish appropriate expression. Expression of the GDF-11 transgenes,thereby decreasing the GDF-11 in the tissue and serum levels of thetransgenic animals and consequently increasing the muscle tissue contentresults in the foodstuffs from these animals (i.e. eggs, beef, pork,poultry meat, milk, etc.) having markedly increased muscle content, andpreferably without increased, and more preferably, reduced levels of fatand cholesterol. By practice of the subject invention, a statisticallysignificant increase in muscle content, preferably at least a 2%increase in muscle content (e.g., in chickens), more preferably a 25%increase in muscle content as a percentage of body weight, morepreferably greater than 40% increase in muscle content in thesefoodstuffs can be obtained.

[0121] In addition decrease in GDF-11 in the tissue and serum levels ofthe transgenic animals can be used to increase the bone content ofanimals use as foodstuffs, for example the transgenic animals havingreduced GDF-11 can be provided to have an additional number of ribs.

[0122] Additional Methods of Use

[0123] Thus, the present invention includes methods for increasingmuscle mass and/or rib content in domesticated animals, characterized byinactivation or deletion of the gene encoding growth and differentiationfactor-11 (GDF-11). The domesticated animal is preferably selected fromthe group consisting of ovine, bovine, porcine, piscine and avian. Theanimal may be treated with an isolated polynucleotide sequence encodinggrowth and differentiation factor-11 which polynucleotide sequence isalso from a domesticated animal selected from the group consisting ofovine, bovine, porcine, piscine and avian. The present inventionincludes methods for increasing the muscle mass or rib content indomesticated animals characterized by administering to a domesticatedanimal monoclonal antibodies directed to the GDF-11 polypeptide. Theantibody may be an anti-GDF-11, and may be either a monoclonal antibodyor a polyclonal antibody.

[0124] The invention includes methods comprising using an anti-GDF-11monoclonal antibody, antisense, or dominant negative mutants as atherapeutic agent to inhibit the growth regulating actions of GDF-11 onmuscle cells. Muscle cells are defined to include fetal or adult musclecells, as well as progenitor cells which are capable of differentiationinto muscle. The monoclonal antibody may be a humanized (e.g., eitherfully or a chimeric) monoclonal antibody, of any species origin, such asmurine, ovine, bovine, porcine or avian. Methods of producing antibodymolecules with various combinations of “humanized” antibodies are wellknown in the art and include combining murine variable regions withhuman constant regions (Cabily, et al. Proc.Natl.Acad.Sci. USA, 81:3273,1984), or by grafting the murine-antibody complementary determiningregions (CDRs) onto the human framework (Richmann, et al., Nature332:323, 1988). Other general references which teach methods forcreating humanized antibodies include Morrison, et al., Science,229:1202, 1985; Jones, et al., Nature, 321:522, 1986; Monroe, et al.,Nature 312:779, 1985; Oi, et al., BioTechniques, 4:214, 1986; EuropeanPatent Application No. 302,620; and U.S. Pat. No. 5,024,834. Therefore,by humanizing the monoclonal antibodies of the invention for in vivouse, an immune response to the antibodies would be greatly reduced.

[0125] The invention includes methods comprising using an anti-GDF-11monoclonal antibody, antisense, or dominant negative mutants as atherapeutic agent to inhibit the growth regulating actions of GDF-11 onbone cells. bone cells are defined to include fetal or adult bone cells,as well as progenitor cells which are capable of differentiation intobone. The monoclonal antibody may be a humanized (e.g., either fully ora chimeric) monoclonal antibody, of any species origin, such as murine,ovine, bovine, porcine or avian. Methods of producing antibody moleculeswith various combinations of “humanized” antibodies are well known inthe art and include combining murine variable regions with humanconstant regions (Cabily, et al. Proc.Natl.Acad.Sci. USA, 81 3273,1984), or by grafting the murine-antibody complementary determiningregions (CDRs) onto the human framework (Richmann, et al., Nature332:323, 1988). Other general references which teach methods forcreating humanized antibodies include Morrison, et al., Science,229:1202, 1985; Jones, et al., Nature, 321:522, 1986; Monroe, et al.,Nature 312:779, 1985; Oi, et al., BioTechniques, 4:214, 1986; EuropeanPatent Application No. 302,620; and U.S. Pat. No. 5,024,834. Therefore,by humanizing the monoclonal antibodies of the invention for in vivouse, an immune response to the antibodies would be greatly reduced.

[0126] The monoclonal antibody, GDF-11 polypeptide, or GDF-11polynucleotide (all “GDF-11 agents”) may have the effect of increasingthe development of skeletal muscles or skeletal bones. In preferredembodiments of the claimed methods, the GDF-11 monoclonal antibody,polypeptide, or polynucleotide is administered to a patient sufferingfrom a disorder selected from the group consisting of muscle wastingdisease, neuromuscular disorder, muscle atrophy or aging. In anotherpreferred embodiment the invention provides a method for treating bonedegenerative disorders, such as osteoporosis by administering to apatient suffering from a disorder antibodies, polypeptides orpolynucleotides effecting GDF-11 activity. The GDF-11 agent may also beadministered to a patient suffering from a disorder selected from thegroup consisting of muscular dystrophy, spinal cord injury, traumaticinjury, congestive obstructive pulmonary disease (COPD), AIDS orcachechia.

[0127] In a preferred embodiment, the GDF-11 agent is administered to apatient with muscle or bone wasting disease or disorder by intravenous,intramuscular or subcutaneous injection; preferably, a monoclonalantibody is administered within a dose range between about 0.1 mg/kg toabout 100 mg/kg; more preferably between about 1 ug/kg to 75 mg/kg; mostpreferably from about 10 mg/kg to 50 mg/kg. The antibody may beadministered, for example, by bolus injunction or by slow infusion. Slowinfusion over a period of 30 minutes to 2 hours is preferred. The GDF-11agent may be formulated in a formulation suitable for administration toa patient. Such formulations are known in the art.

[0128] The dosage regimen will be determined by the attending physicianconsidering various factors which modify the action of the GDF-11protein, e.g. amount of tissue desired to be formed, the site of tissuedamage, the condition of the damaged tissue, the size of a wound, typeof damaged tissue, the patient's age, sex, and diet, the severity of anyinfection, time of administration and other clinical factors. The dosagemay vary with the type of matrix used in the reconstitution and thetypes of agent, such as anti-GDF-11 antibodies, to be used in thecomposition. Generally, systemic or injectable administration, such asintravenous (IV), intramuscular (IM) or subcutaneous (Sub-Q) injection.Administration will generally be initiated at a dose which is minimallyeffective, and the dose will be increased over a preselected time courseuntil a positive effect is observed. Subsequently, incremental increasesin dosage will be made limiting such incremental increases to suchlevels that produce a corresponding increase in effect, while takinginto account any adverse affects that may appear. The addition of otherknown growth factors, such as IGF I (insulin like growth factor I),human, bovine, or chicken growth hormone which may aid in increasingmuscle mass, to the final composition, may also affect the dosage. Inthe embodiment where an anti-GDF-11 antibody is administered, theanti-GDF-11 antibody is generally administered within a dose range ofabout 0.1 ug/kg to about 100 mg/kg.; more preferably between about 10mg/kg to 50 mg/kg.

[0129] Progress can be monitored by periodic assessment of tissue growthand/or repair. The progress can be monitored, for example, x-rays,histomorphometric determinations and tetracycline labeling.

[0130] Screening for GDF-11 Modulating Compounds

[0131] In another embodiment, the invention provides a method foridentifying a compound or molecule that modulates GDF-11 proteinactivity or gene expression. The method includes incubating componentscomprising the compound, GDF-11 polypeptide or with a recombinant cellexpressing GDF-11 polypeptide, under conditions sufficient to allow thecomponents to interact and determining the effect of the compound onGDF-11 activity or expression. The effect of the compound on GDF-11activity can be measured by a number of assays, and may includemeasurements before and after incubating in the presence of thecompound. Compounds that affect GDF-11 activity or gene expressioninclude peptides, peptidomimetics, polypeptides, chemical compounds andbiologic agents. Assays include Northern blot analysis of GDF-11 mRNA(for gene expression), Western blot analysis (for protein level) andmuscle fiber analysis (for protein activity).

[0132] The above screening assays may be used for detecting thecompounds or molecules that bind to the GDF-11 receptor or GDF-11polypeptide, in isolating molecules that bind to the GDF-11 gene, formeasuring the amount of GDF-11 in a sample, either polypeptide or RNA(mRNA), for identifying molecules that may act as agonists orantagonists, and the like. For example, GDF-11 antagonists are usefulfor treatment of muscular and adipose tissue disorders (e.g., obesity).

[0133] Incubating includes conditions which allow contact between thetest compound and GDF-11 polypeptide or with a recombinant cellexpressing GDF-11 polypeptide. Contacting includes in solution and insolid phase, or in a cell. The test compound may optionally be acombinatorial library for screening a plurality of compounds. Compoundsidentified in the method of the invention can be further evaluated,detected, cloned, sequenced, and the like, either in solution or afterbinding to a solid support, by any method usually applied to thedetection of a specific DNA sequence such as PCR, oligomer restriction(Saiki, et al., Bio/Technology, 3:1008-1012, 1985), allele-specificoligonucleotide (ASO) probe analysis (Conner, et al., Proc. Natl. Acad.Sci. USA, 80:278, 1983), oligonucleotide Landegren, et al., Science,241:1077, 1988), and the like. Molecular techniques for DNA analysishave been reviewed (Landegren, et al., Science, 242:229-237, 1988).

[0134] All references cited herein are hereby incorporated by referencein their entirety.

[0135] The following examples are intended to illustrate but not limitthe invention. While they are typical of those that might be used, otherprocedures known to those skilled in the art may alternatively be used.

EXAMPLE 1 Identification and Isolation of a Novel TGF-β Family Member

[0136] To identify novel members of the TGF-β superfamily, a murinegenomic library was screened at reduced stringency using a murine GDF-8probe (FIG. 8; nucleotides 865-1234) spanning the region encoding theC-terminal portion of the GDF-8 precursor protein. Hybridization wascarried out as described (Lee, Mol. Endocrinol., 4:1034, 1990) at 65°C., and the final wash was carried out at the same temperature in abuffer containing 0.5 M NaCl. Among the hybridizing phage was one thatcould be distinguished from GDF-8-containing phage on the basis of itsreduced hybridization intensity to the GDF-8 probe. Partial nucleotidesequence analysis of the genomic insert present in this weaklyhybridizing phage showed that this clone contained a sequence highlyrelated to but distinct from murine GDF-8.

[0137] A partial nucleotide sequence of the genomic insert present inthis phage is shown in FIG. 1a. The sequence contained an open readingframe extending from nucleotides 198 to 575 that showed significanthomology to the known members of the TGF-β superfamily (see below).Preceding this sequence was a 3′ splice consensus sequence at preciselythe same position as in the GDF-8 gene. This new TGF-β family member wasgiven the designation GDF-11 (growth/differentiation factor-11).

EXAMPLE 2 Expression of GDF-11

[0138] To determine the expression pattern of GDF-11, RNA samplesprepared from a variety of tissues were screened by Northern analysis.RNA isolation and Northern analysis were carried out as describedpreviously (Lee, Mol. Endocrinol., 4:1034, 1990) except that thehybridization was carried out in 5×SSPE, 10% dextran sulfate, 50%formamide, 1% SDS, 200 μg/ml salmon DNA, and 0.1% each of bovine serumalbumin, ficoll, and polyvinylpyrrolidone. Five micrograms of twice polyA-selected RNA prepared from each tissue (except for 2 day neonatalbrain, for which only 3.3 μg RNA were used) were electrophoresed onformaldehyde gels, blotted, and probed with GDF-11. As shown in FIG. 2,the GDF-11 probe detected two RNA species, approximately 4.2 and 3.2 kbin length, in adult thymus, brain, spleen, uterus, and muscle as well asin whole embryos isolated at day 12.5 or 18.5 and in brain samples takenat various stages of development. On longer exposures of these blots,lower levels of GDF-11 RNA could also be detected in a number of othertissues.

EXAMPLE 3 Isolation of cDNA Clones Encoding GDF-11

[0139] In order to isolate cDNA clones encoding GDF-11, a cDNA librarywas prepared in the lambda ZAP II vector (Stratagene) using RNA preparedfrom human adult spleen. From 5 μg of twice poly A-selected RNA preparedfrom human spleen, a cDNA library consisting of 21 million recombinantphage was constructed according to the instructions provided byStratagene. The library was screened without amplification. Libraryscreening and characterization of cDNA inserts were carried out asdescribed previously (Lee, Mol. Endocrinol., 4:1034, 1990). From thislibrary, 23 hybridizing phage were obtained.

[0140] The entire nucleotide sequence of the clone extending furthesttoward the 5′ end of the gene was determined. The 1258 base pairsequence contained a single long open reading frame beginning from the5′ end of the clone and extending to a TAA stop codon. Because the openreading frame and the homology with GDF-8 (see below) extended to thevery 5′ end of the clone, it seemed likely that this clone was missingthe coding sequence corresponding to the N-terminal portion of theGDF-11 precursor protein. In order to obtain the remaining portion ofthe GDF-11 sequence, several genomic clones were isolated by screening ahuman genomic library with the human GDF-11 cDNA probe. Partial sequenceanalysis of one of these genomic clones showed that this clone containedthe GDF-11 gene. From this clone, the remaining GDF-11 coding sequencewas obtained. FIG. 1b shows the predicted sequence of GDF-11 assembledfrom the genomic and cDNA sequences. Nucleotides 136 to 1393 representthe extent of the sequence obtained from a cDNA clone. Nucleotides 1 to135 were obtained from a genomic clone. The sequence has beenarbitrarily numbered beginning with a Sac II site present in the genomicclone, but the location of the mRNA start site is not known. Thesequence contains a putative initiating methionine at nucleotide 54.Whether the sequence upstream of this methionine codon is all present inthe mRNA is not known. Beginning with this methionine codon, the openreading frame extends for 407 amino acids. The sequence contains onepotential N-linked glycosylation site at asparagine 94. The sequencecontains a predicted RXXR proteolytic cleavage site at amino acids 295to 298, and cleavage of the precursor at this site would generate anactive C-terminal fragment 109 amino acids in length with a predictedmolecular weight of approximately 12,500 kD. In this region, thepredicted murine and human GDF-11 amino acid sequences are 100%identical. The high degree of sequence conservation across speciessuggests that GDF-11 plays an important role in vivo.

[0141] The C-terminal region following the predicted cleavage sitecontains all the hallmarks present in other TGF-β family members. GDF-11contains most of the residues that are highly conserved in other familymembers, including the seven cysteine residues with their characteristicspacing. Like the TGF-β's, the inhibin β's, and GDF-8, GDF-11 alsocontains two additional cysteine residues. In the case of TGF-β2, theseadditional cysteine residues are known to form an intramoleculardisulfide bond (Daopin, et al., Science, 257:369, 1992; Schlunegger andGrutter, Nature, 358:430, 1992). A tabulation of the amino acid sequencehomologies between GDF-11 and the other TGF-β family members is shown inFIG. 3. Numbers represent percent amino acid identities between eachpair calculated from the first conserved cysteine to the C-terminus.Boxes represent homologies among highly-related members withinparticular subgroups. In this region, GDF-11 is most highly related toGDF-8 (92% sequence identity).

[0142] An alignment of GDF-8 (SEQ ID NO:5) and GDF-11 (SEQ ID NO:6)amino acid sequences is shown in FIG. 4a. The two sequences containpotential N-linked glycosylation signals (NIS) and putative proteolyticprocessing sites (RSRR) at analogous positions. The two sequences arerelated not only in the C-terminal region following the putativecleavage site (90% amino acid sequence identity), but also in thepro-region of the molecules (45% amino acid sequence identity).

EXAMPLE 4 Construction of a Hybrid GDF-8/GDF11 Gene

[0143] In order to express GDF-11 protein, a hybrid gene was constructedin which the N-terminal region of GDF-11 was replaced by the analogousregion of GDF-8. Such hybrid constructs have been used to producebiologically-active BMP-4 (Hammonds, et al., Mol. Endocrinol., 5:149,1991) and Vg-1 (Thomsen and Melton, Cell, 74:433, 1993). In order toensure that the GDF-11 protein produced from the hybrid construct wouldrepresent authentic GDF-11, the hybrid gene was constructed in such amanner that the fusion of the two gene fragments would occur preciselyat the predicted cleavage sites. In particular, an AvaII restrictionsite is present in both sequences at the location corresponding to thepredicted proteolytic cleavage site. The N-terminal pro-region of GDF-8up to this AvaII site was obtained by partial digestion of the clonewith AvaII and fused to the C-terminal region of GDF-11 beginning atthis AvaII site. The resulting hybrid construct was then inserted intothe pMSXND mammalian expression vector (Lee and Nathans, J. Biol. Chem.,263:3521) and transfected into Chinese hamster ovary cells. As shown inFIG. 5, Western analysis of conditioned medium from G418-resistant cellsusing antibodies raised against the C-terminal portion of GDF-8 showedthat these cells secreted GDF-11 protein into the medium and that atleast some of the hybrid protein was proteolytically processed.Furthermore, these studies demonstrate that the antibodies directedagainst the C-terminal portion of GDF-8 will also react with GDF-11protein.

EXAMPLE 5 Chromosomal Localization of GDF-11

[0144] In order to map the chromosomal location of GDF-11, DNA samplesfrom human/rodent somatic cell hybrids (Drwinga, et al., Genomics,16:311-313, 1993; Dubois and Naylor, Genomics, 16:315-319, 1993) wereanalyzed by polymerase chain reaction followed by Southern blotting.Polymerase chain reaction was carried out using primer #101,5′-GAGTCCCGCTGCTGCCGATATCC-3′, (SEQ ID NO:7) and primer #102,5′-TAGAGCATGTTGATTGGGGACAT-3′, (SEQ ID NO:8) for 35 cycles at 94° C. for2 minutes, 58° C. for 1 minutes, and 72° C. for 1 minute. These primerscorrespond to nucleotides 981 to 1003 and the reverse complement ofnucleotides 1182 to 1204, respectively, in the human GDF-11 sequence.PCR products were electrophoresed on agarose gels, blotted, and probedwith oligonucleotide #104, 5′-AAATATCCGCATACCCATTT-3′, (SEQ ID NO:9)which corresponds to a sequence internal to the region flanked by primer#101 and #102. Filters were hybridized in 6×SSC, 1× Denhardt's solution,100 μg/ml yeast transfer RNA, and 0.05% sodium pyrophosphate at 50° C.

[0145] As shown in FIG. 6, the human-specific probe detected a band ofthe predicted size (approximately 224 base pairs) in the positivecontrol sample (total human genomic DNA) and in a single DNA sample fromthe human/rodent hybrid panel. This positive signal corresponds to humanchromosome 12. The human chromosome contained in each of the hybrid celllines is identified at the top of each of the first 24 lanes (1-22, X,and Y). In the lanes designated CHO, M, and H, the starting DNA templatewas total genomic DNA from hamster, mouse, and human sources,respectively. In the lane marked B1, no template DNA was used. Numbersat left indicate the mobilities of DNA standards. These data show thatthe human GDF-11 gene is located on chromosome 12.

[0146] In order to determine the more precise location of GDF-11 onchromosome 12, the GDF-11 gene was localized by florescence in situhybridization (FISH). These FISH localization studies were carried outby contract to BIOS laboratories (New Haven, Conn.). Purified DNA from ahuman GDF-11 genomic clone was labelled with digoxigenin dUTP by nicktranslation. Labelled probe was combined with sheared human DNA andhybridized to normal metaphase chromosomes derived from PHA stimulatedperipheral blood lymphocytes in a solution containing 50% formamide, 10%dextran sulfate and 2×SSC. Specific hybridization signals were detectedby incubating the hybridized slides in fluorescein-conjugated sheepantidigoxigenin antibodies. Slides were then counterstained withpropidium iodide and analyzed. As shown in FIG. 7a, this experimentresulted in the specific labelling of the proximal long arm of a group Cchromosome, the size and morphology of which were consistent withchromosome 12. In order to confirm the identity of the specificallylabelled chromosome, a second experiment was conducted in which achromosome 12-specific centromere probe was cohybridized with GDF-11. Asshown in FIG. 7b, this experiment clearly demonstrated that GDF-11 islocated at a position which is 23% of the distance from the centromereto the telomere of the long arm of chromosome 12, an area whichcorresponds to band 12q13 (FIG. 7c). A total of 85 metaphase cells wereanalyzed and 80 exhibited specific labelling.

EXAMPLE 6 GDF-11 Homology in Mammalian Species

[0147] Like most other TGF-β family member, GDF-11 also appears to behighly conserved across species. By genomic Southern analysis,homologous sequences were detected in all mammalian species examined aswell as in chickens and frogs (FIG. 4b). In most species, the GDF-11probe also detected a second, more faintly hybridizing fragmentcorresponding to the myostatin gene (McPherron et al., 1997).

EXAMPLE 7 GDF-11 Transgenic Knockout Mice

[0148] To determine the biological function of GDF-11, we disrupted theGDF-11 gene by homologous targeting in embryonic stem cells. A murine129 SV/J genomic library was prepared in lambda FIXII according to theinstructions provided by Stratagene (La Jolla, Calif.). The structure ofthe GDF-11 gene was deduced from restriction mapping and partialsequencing of phage clones isolated from the library. Vectors forpreparing the targeting construct were kindly provided by Philip Sorianoand Kirk Thomas. To ensure that the resulting mice would be null forGDF-11 function, the entire mature C-terminal region was deleted andreplaced by a neo cassette (FIGS. 9a,b). R1 ES cells were transfectedwith the targeting construct, selected with gancyclovir (2 μM) and G418(250 μg/ml), and analyzed by Southern analysis. Homologous targeting ofthe GDF-11 gene was seen in 8/155 gancyclovir/G418 doubly resistant EScell clones. Following injection of several targeted clones intoC57BL/6J blastocysts, we obtained chimeras from one ES clone thatproduced heterozygous pups when crossed to both C57BL/6J and 129/SvJfemales. Crosses of C57BL/6J/129/SvJ hybrid F1 heterozygotes produced 49wild-type (34%), 94 heterozygous (66%) and no homozygous mutant adultoffspring. Similarly, there were no adult homozygous null animals seenin the 129/SvJ background (32 wild-type (36%) and 56 heterozygous mutant(64%) animals).

[0149] To determine the age at which homozygous mutants were dying, wegenotyped litters of embryos isolated at various gestational ages fromheterozygous females that had been mated to heterozygous males. At allembryonic stages examined, homozygous mutant embryos were present atapproximately the predicted frequency of 25%. Among hybrid newborn mice,the different genotypes were also represented at the expected Mendelianratio of 1:2:1 (34 +/+ (28%), 61 +/− (50%), and 28 −/− (23%)).Homozygous mutant mice were born alive and were able to breath andnurse. All homozygous mutants died, however, within the first 24 hoursafter birth. The precise cause of death was unknown, but the lethalitymay have been related to the fact that the kidneys in homozygous mutantswere either severely hypoplastic or completely absent. A summary of thekidney abnormalities in these mice is shown in FIG. 10.

EXAMPLE 8 Anatomical Differences in Knockout Mice

[0150] Homozygous mutant animals were easily recognizable by theirseverely shortened or absent tails (FIG. 11a). To further characterizethe tail defects in these homozygous mutant animals, we examined theirskeletons to determine the degree of disruption of the caudal vertebrae.A comparison of wild-type and mutant skeleton preparations of late stageembryos and newborn mice, however, revealed differences not only in thecaudal region of the animals but in many other regions as well. Innearly every case where differences were noted, the abnormalitiesappeared to represent homeotic transformations of vertebral segments inwhich particular segments appeared to have a morphology typical of moreanterior segments. These transformations, which are summarized in FIG.12, were evident throughout the axial skeleton extending from thecervical region to the caudal region. Except for the defects seen in theaxial skeleton, the rest of the skeleton, such as the cranium and limbbones, appeared normal.

[0151] Anterior transformations of the vertebrae in mutant newbornanimals were most readily apparent in the thoracic region, where therewas a dramatic increase in the number of thoracic (T) segments. Allwild-type mice examined showed the typical pattern of 13 thoracicvertebrae each with its associated pair of ribs (FIGS. 11(b,e)). Incontrast, homozygous mutant mice showed a striking increase in thenumber of thoracic vertebrae. All homozygous mutants examined had 4 to 5extra pairs of ribs for a total of 17 to 18 (FIGS. 11(d,g)) although inover ⅓ of these animals, the 18th rib appeared to be rudimentary. Hence,segments that would normally correspond to lumbar (L) segments L1 to L4or L5 appeared to have been transformed into thoracic segments in mutantanimals.

[0152] Moreover, transformations within the thoracic region in which onethoracic vertebra had a morphology characteristic of another thoracicvertebra were also evident. For example, in wild-type mice, the first 7pairs of ribs attach to the sternum, and the remaining 6 are unattachedor free (FIGS. 11(e,h)). In homozygous mutants, there was an increase inthe number of both attached and free pairs of ribs to 10-11 and 7-8,respectively (FIGS. 11(g,j)). Therefore, thoracic segments T8, T9, T10,and in some cases even T11, which all have free ribs in wild-typeanimals, were transformed in mutant animals to have a characteristictypical of more anterior thoracic segments, namely, the presence of ribsattached to the sternum. Consistent with this finding, the transitionalspinous process and transitional articular processes which are normallyfound on T10 in wild-type animals were instead found on T13 inhomozygous mutants (data not shown). Additional transformations withinthe thoracic region were also noted in certain mutant animals. Forexample, in wild-type mice, the ribs derived from T1 normally touch thetop of the sternum. However, in {fraction (2/23)} hybrid and ⅔ 129/SvJhomozygous mutant mice examined, T2 appeared to have been transformed tohave a morphology resembling that of T1; that is, in these animals, theribs derived from T2 extended to touch the top of the sternum. In thesecases, the ribs derived from T1 appeared to fuse to the second pair ofribs. Finally, in 82% of homozygous mutants, the long spinous processnormally present on T2 was shifted to the position of T3. In certainother homozygous mutants, asymmetric fusion of a pair of vertebrostemalribs was seen at other thoracic levels.

[0153] The anterior transformations were not restricted to the thoracicregion. The anterior most transformation that we observed was at thelevel of the 6th cervical vertebra (C6). In wild-type mice, C6 isreadily identifiable by the presence of two anterior tuberculi on theventral side. In several homozygous mutant mice, although one of thesetwo anterior tuberculi was present on C6, the other was present at theposition of C7 instead. Hence, in these mice, C7 appeared to have beenpartially transformed to have a morphology resembling that of C6. Oneother homozygous mutant had 2 anterior tuberculi on C7 but retained oneon C6 for a complete C7 to C6 transformation but a partial C6 to C5transformation.

[0154] Transformations of the axial skeleton also extended into thelumbar region. Whereas wild-type animals normally have only 6 lumbarvertebrae, homozygous mutants had 8-9. At least 6 of the lumbarvertebrae in the mutants must have derived from segments that wouldnormally have given rise to sacral and caudal vertebrae as the datadescribed above suggest that 4 to 5 lumbar segments were transformedinto thoracic segments. Hence, homozygous mutant mice had a total of33-34 presacral vertebrae compared to 26 presacral vertebrae normallypresent in wild-type mice. The most common presacral vertebral patternswere C7/T18/L8 and C7/T18/L9 for mutant mice compared to C7/T13/L6 forwild-type mice. The presence of additional presacral vertebrae in mutantanimals was obvious even without detailed examination of the skeletonsas the position of the hindlimbs relative to the forelimbs was displacedposteriorly by 7-8 segments.

[0155] Although the sacral and caudal vertebrae were also affected inhomozygous mutant mice, the exact nature of each transformation was notas readily identifiable. In wild-type mice, sacral segments S1 and S2typically have broad transverse processes compared to S3 and S4. In themutants, there did not appear to be an identifiable S1 or S2 vertebra.Instead, mutant animals had several vertebrae that appeared to havemorphology similar to S3. In addition, the transverse processes of all 4sacral vertebrae are normally fused to each other although in newbornsoften only fusions of the first 3 vertebrae are seen. In homozygousmutants, however, the transverse processes of the sacral vertebrae wereusually unfused. In the caudalmost region, all mutant animals also hadseverely malformed vertebrae with extensive fusions of cartilage.Although the severity of the fusions made it difficult to count thetotal number of vertebrae in the caudal region, we were able to count upto 15 transverse processes in several animals. We were unable todetermine whether these represented sacral or caudal vertebrae in themutants because we could not establish morphologic criteria fordistinguishing S4 from caudal vertebrae even in wild-type newbornanimals. Regardless of their identities, the total number of vertebraein this region was significantly reduced from the normal number ofapproximately 30. Hence, although the mutants had significantly morethoracic and lumber vertebrae than wild-type mice, the total number ofsegments was reduced in the mutants due to the truncation of the tails.

[0156] Heterozygous mice also showed abnormalities in the axial skeletonalthough the phenotype was much milder than in homozygous mice. The mostobvious abnormality in heterozygous mice was the presence of anadditional thoracic segment with an associated pair of ribs (FIGS.11(c,f)). This transformation was present in every heterozygous animalexamined, and in every case, the additional pair of ribs was attached tothe sternum (FIG. 11(I)). Hence, T8, whose associated rib normally doesnot touch the sternum, appeared to have been transformed to a morphologycharacteristic of a more anterior thoracic vertebra, and L1 appeared tohave been transformed to a morphology characteristic of a posteriorthoracic vertebra. Other abnormalities indicative of anteriortransformations were also seen to varying degrees in heterozygous mice.These included a shift of the long spinous process characteristic of T2by one segment to T3, a shift of the articular and spinous processesfrom T10 to T11, a shift of the anterior tuberculus on C6 to C7, andtransformation of T2 to T1 where the rib associated with T2 touched thetop of the sternum.

[0157] In order to understand the basis for the abnormalities in axialpatterning seen in GDF-11 mutant mice, we examined mutant embryosisolated at various stages of development and compared them to wild-typeembryos. By gross morphological examination, homozygous mutant embryosisolated up to day 9.5 of gestation were not readily distinguishablefrom corresponding wild-type embryos. In particular, the number ofsomites present at any given developmental age was identical betweenmutant and wild-type embryos, suggesting that the rate of somiteformation was unaltered in the mutants. By day 10.5-11.5 p.c., mutantembryos could be easily distinguished from wild-type embryos by theposterior displacement of the hindlimb by 7-8 somites. The abnormalitiesin tail development were also readily apparent at this stage. Takentogether, these data suggest that the abnormalities observed in themutant skeletons represented true transformations of segment identitiesrather than the insertion of additional segments, for example, by anenhanced rate of somitogenesis.

[0158] Alterations in expression of homeobox containing genes are knownto cause transformations in Drosophila and in vertebrates. To see if theexpression patterns of Hox genes (the vertebrate homeobox containinggenes) were altered in GDF-11 null mutants we determined the expressionpattern of 3 representative Hox genes, Hoxc-6, Hoxc-8 and Hoxc-11, inday 12.5 p.c. wild-type, heterozygous and homozygous mutant embryos bywhole mount in situ hybridization. The expression pattern of Hoxc-6 inwild-type embryos spanned prevertebrae 8-15 which correspond to thoracicsegments T1-T8. In homozygous mutants, however, the Hoxc-6 expressionpattern was shifted posteriorly and expanded to prevertebrae 9-18(T2-T11). A similar shift was seen with the Hoxc-8 probe. In wild-typeembryos, Hoxc-8 was expressed in prevertebrae 13-18 (T6-T11) but, inhomozygous mutant embryos, Hoxc-8 was expressed in prevertebrae 14-22(T7-T15). Finally, Hoxc-11 expression was also shifted posteriorly inthat the anterior boundary of expression changed from prevertebrae 28tin wild-type embryos to prevertebrae 36 in mutant embryos. (Note thatbecause the position of the hindlimb is also shifted posteriorly inmutant embryos, the Hoxc-11 expression patterns in wild-type and mutantappeared similar relative to the hindlimbs). These data provide furtherevidence that the skeletal abnormalities seen in mutant animalsrepresent homeotic transformations.

[0159] The phenotype of GDF-11 mice suggested that GDF-11 acts earlyduring embryogenesis as a global regulator of axial patterning. To beginto examine the mechanism by which GDF-11 exerts its effects, wedetermined the expression pattern of GDF-11 in early mouse embryos bywhole mount in situ hybridization. At these stages the primary sites ofGDF-11 expression correlated precisely with the known sites at whichmesodermal cells are generated. Expression of GDF-11 was first detectedat day 8.25-8.5 p.c. (8-10 somites) in the primitive streak region,which is the site at which ingressing cells form the mesoderm of thedeveloping embryo. Expression was maintained in the primitive streak atday 8.75, but by day 9.5 p.c., when the tail bud replaces the primitivestreak as the source of new mesodermal cells, expression of GDF-11shifted to the tail bud. Hence at these early stages, GDF-11 appears tobe synthesized in the region of the developing embryo where newmesodermal cells arise and presumably acquire their positional identity.

[0160] The phenotype of GDF-11 knockout mice in several respectsresembles the phenotype of mice carrying a deletion of a receptor forsome members of the TGF-β superfamily, the activin type IIB receptor(ActRIIB). As in the case of GDF-11 knockout mice, the ActRIIB knockoutmice have extra pairs of ribs and a spectrum of kidney defects rangingfrom hypoplastic kidneys to complete absence of kidneys. The similarityin the phenotypes of these mice raises the possibility that ActRIIB maybe a receptor for GDF-11. However, Act RIIB cannot be the sole receptorfor GDF-11 Ibecause the phenotype of GDF-11 knockout mice is more severethan the phenotype of ActRIIB mice. For example, whereas the GDF-11knockout animals have 4-5 extra pairs of ribs and show homeotictransformations throughout the axial skeleton, the ActRIIB knockoutanimals have only 3 extra pairs of ribs and do not show transformationsat other axial levels. In addition, the data indicate that the kidneydefects in the GDF-11 knockout mice are also more severe than those inActRIIB knockout mice. The ActRIIB knockout mice show defects inleft/right axis formation, such as lung isomerixm and a range of heartdefects that we have not yet observed in GDF-11 knockout mice. ActRIIBcan bind the activins and certain BMPs, although none of the knockoutmice generated for these ligands show defects in left/right axisformation.

[0161] If GDF-11 does act directly on mesodermal cells to establishpositional identity, the data presented here would be consistent witheither short range or morphogen models for GDF-11 action. That is,GDF-11 may act on mesodermal precursors to establish patterns of Hoxgene expression as these cells are being generated at the site of GDF-11expression, or alternatively, GDF-11 produced at the posterior end ofthe embryo may diffuse to form a morphogen gradient. Whatever themechanism of action of GDF-11 may be, the fact that grossanterior/posterior patterning still does occur in GDF-11 knockoutanimals suggests that GDF-11 may not be the sole regulator ofanterior/posterior specification. Nevertheless, it is clear that GDF-11plays an important role as a global regulator of axial patterning andthat further study of this molecule will lead to important new insightsinto how positional identity along the anterior/posterior axis isestablished in the vertebrate embryo.

[0162] Although the invention has been described with reference to thepresently preferred embodiment, it should be understood that variousmodifications can be made without departing from the spirit of theinvention. Accordingly, the invention is limited only by the followingclaims.

1 10 1 1393 DNA Human GDF-11 CDS (54)..(1274) 1 ccgcgggact ccggcgtccccgccccccag tcctccctcc cctcccctcc agc atg 56 Met 1 gtg ctc gcg gcc ccgctg ctg ctg ggc ttc ctg ctc ctc gcc ctg gag 104 Val Leu Ala Ala Pro LeuLeu Leu Gly Phe Leu Leu Leu Ala Leu Glu 5 10 15 ctg cgg ccc cgg ggg gaggcg gcc gag ggc ccc gcg gcg gcg gcg gcg 152 Leu Arg Pro Arg Gly Glu AlaAla Glu Gly Pro Ala Ala Ala Ala Ala 20 25 30 gcg gcg gcg gcg gcg gca gcggcg ggg gtc ggg ggg gag cgc tcc agc 200 Ala Ala Ala Ala Ala Ala Ala AlaGly Val Gly Gly Glu Arg Ser Ser 35 40 45 cgg cca gcc ccg tcc gtg gcg cccgag ccg gac ggc tgc ccc gtg tgc 248 Arg Pro Ala Pro Ser Val Ala Pro GluPro Asp Gly Cys Pro Val Cys 50 55 60 65 gtt tgg cgg cag cac agc cgc gagctg cgc cta gag agc atc aag tcg 296 Val Trp Arg Gln His Ser Arg Glu LeuArg Leu Glu Ser Ile Lys Ser 70 75 80 cag atc ttg agc aaa ctg cgg ctc aaggag gcg ccc aac atc agc cgc 344 Gln Ile Leu Ser Lys Leu Arg Leu Lys GluAla Pro Asn Ile Ser Arg 85 90 95 gag gtg gtg aag cag ctg ctg ccc aag gcgccg ccg ctg cag cag atc 392 Glu Val Val Lys Gln Leu Leu Pro Lys Ala ProPro Leu Gln Gln Ile 100 105 110 ctg gac cta cac gac ttc cag ggc gac gcgctg cag ccc gag gac ttc 440 Leu Asp Leu His Asp Phe Gln Gly Asp Ala LeuGln Pro Glu Asp Phe 115 120 125 ctg gag gag gac gag tac cac gcc acc accgag acc gtc att agc atg 488 Leu Glu Glu Asp Glu Tyr His Ala Thr Thr GluThr Val Ile Ser Met 130 135 140 145 gcc cag gag acg gac cca gca gta cagaca gat ggc agc cct ctc tgc 536 Ala Gln Glu Thr Asp Pro Ala Val Gln ThrAsp Gly Ser Pro Leu Cys 150 155 160 tgc cat ttt cac ttc agc ccc aag gtgatg ttc aca aag gta ctg aag 584 Cys His Phe His Phe Ser Pro Lys Val MetPhe Thr Lys Val Leu Lys 165 170 175 gcc cag ctg tgg gtg tac cta cgg cctgta ccc cgc cca gcc aca gtc 632 Ala Gln Leu Trp Val Tyr Leu Arg Pro ValPro Arg Pro Ala Thr Val 180 185 190 tac ctg cag atc ttg cga cta aaa ccccta act ggg gaa ggg acc gca 680 Tyr Leu Gln Ile Leu Arg Leu Lys Pro LeuThr Gly Glu Gly Thr Ala 195 200 205 ggg gga ggg ggc gga ggc cgg cgt cacatc cgt atc cgc tca ctg aag 728 Gly Gly Gly Gly Gly Gly Arg Arg His IleArg Ile Arg Ser Leu Lys 210 215 220 225 att gag ctg cac tca cgc tca ggccat tgg cag agc atc gac ttc aag 776 Ile Glu Leu His Ser Arg Ser Gly HisTrp Gln Ser Ile Asp Phe Lys 230 235 240 caa gtg cta cac agc tgg ttc cgccag cca cag agc aac tgg ggc atc 824 Gln Val Leu His Ser Trp Phe Arg GlnPro Gln Ser Asn Trp Gly Ile 245 250 255 gag atc aac gcc ttt gat ccc agtggc aca gac ctg gct gtc acc tcc 872 Glu Ile Asn Ala Phe Asp Pro Ser GlyThr Asp Leu Ala Val Thr Ser 260 265 270 ctg ggg ccg gga gcc gag ggg ctgcat cca ttc atg gag ctt cga gtc 920 Leu Gly Pro Gly Ala Glu Gly Leu HisPro Phe Met Glu Leu Arg Val 275 280 285 cta gag aac aca aaa cgt tcc cggcgg aac ctg ggt ctg gac tgc gac 968 Leu Glu Asn Thr Lys Arg Ser Arg ArgAsn Leu Gly Leu Asp Cys Asp 290 295 300 305 gag cac tca agc gag tcc cgctgc tgc cga tat ccc ctc aca gtg gac 1016 Glu His Ser Ser Glu Ser Arg CysCys Arg Tyr Pro Leu Thr Val Asp 310 315 320 ttt gag gct ttc ggc tgg gactgg atc atc gca cct aag cgc tac aag 1064 Phe Glu Ala Phe Gly Trp Asp TrpIle Ile Ala Pro Lys Arg Tyr Lys 325 330 335 gcc aac tac tgc tcc ggc cagtgc gag tac atg ttc atg caa aaa tat 1112 Ala Asn Tyr Cys Ser Gly Gln CysGlu Tyr Met Phe Met Gln Lys Tyr 340 345 350 ccg cat acc cat ttg gtg cagcag gcc aat cca aga ggc tct gct ggg 1160 Pro His Thr His Leu Val Gln GlnAla Asn Pro Arg Gly Ser Ala Gly 355 360 365 ccc tgt tgt acc ccc acc aagatg tcc cca atc aac atg ctc tac ttc 1208 Pro Cys Cys Thr Pro Thr Lys MetSer Pro Ile Asn Met Leu Tyr Phe 370 375 380 385 aat gac aag cag cag attatc tac ggc aag atc cct ggc atg gtg gtg 1256 Asn Asp Lys Gln Gln Ile IleTyr Gly Lys Ile Pro Gly Met Val Val 390 395 400 gat cgc tgt ggc tgc tcttaagtgggtc actacaagct gctggagcaa 1304 Asp Arg Cys Gly Cys Ser 405agacttggtg ggtgggtaac ttaacctctt cacagaggat aaaaaatgct tgtgagtatg 1364acagaaggga ataaacaggc ttaaagggt 1393 2 407 PRT Human GDF-11 2 Met ValLeu Ala Ala Pro Leu Leu Leu Gly Phe Leu Leu Leu Ala Leu 1 5 10 15 GluLeu Arg Pro Arg Gly Glu Ala Ala Glu Gly Pro Ala Ala Ala Ala 20 25 30 AlaAla Ala Ala Ala Ala Ala Ala Ala Gly Val Gly Gly Glu Arg Ser 35 40 45 SerArg Pro Ala Pro Ser Val Ala Pro Glu Pro Asp Gly Cys Pro Val 50 55 60 CysVal Trp Arg Gln His Ser Arg Glu Leu Arg Leu Glu Ser Ile Lys 65 70 75 80Ser Gln Ile Leu Ser Lys Leu Arg Leu Lys Glu Ala Pro Asn Ile Ser 85 90 95Arg Glu Val Val Lys Gln Leu Leu Pro Lys Ala Pro Pro Leu Gln Gln 100 105110 Ile Leu Asp Leu His Asp Phe Gln Gly Asp Ala Leu Gln Pro Glu Asp 115120 125 Phe Leu Glu Glu Asp Glu Tyr His Ala Thr Thr Glu Thr Val Ile Ser130 135 140 Met Ala Gln Glu Thr Asp Pro Ala Val Gln Thr Asp Gly Ser ProLeu 145 150 155 160 Cys Cys His Phe His Phe Ser Pro Lys Val Met Phe ThrLys Val Leu 165 170 175 Lys Ala Gln Leu Trp Val Tyr Leu Arg Pro Val ProArg Pro Ala Thr 180 185 190 Val Tyr Leu Gln Ile Leu Arg Leu Lys Pro LeuThr Gly Glu Gly Thr 195 200 205 Ala Gly Gly Gly Gly Gly Gly Arg Arg HisIle Arg Ile Arg Ser Leu 210 215 220 Lys Ile Glu Leu His Ser Arg Ser GlyHis Trp Gln Ser Ile Asp Phe 225 230 235 240 Lys Gln Val Leu His Ser TrpPhe Arg Gln Pro Gln Ser Asn Trp Gly 245 250 255 Ile Glu Ile Asn Ala PheAsp Pro Ser Gly Thr Asp Leu Ala Val Thr 260 265 270 Ser Leu Gly Pro GlyAla Glu Gly Leu His Pro Phe Met Glu Leu Arg 275 280 285 Val Leu Glu AsnThr Lys Arg Ser Arg Arg Asn Leu Gly Leu Asp Cys 290 295 300 Asp Glu HisSer Ser Glu Ser Arg Cys Cys Arg Tyr Pro Leu Thr Val 305 310 315 320 AspPhe Glu Ala Phe Gly Trp Asp Trp Ile Ile Ala Pro Lys Arg Tyr 325 330 335Lys Ala Asn Tyr Cys Ser Gly Gln Cys Glu Tyr Met Phe Met Gln Lys 340 345350 Tyr Pro His Thr His Leu Val Gln Gln Ala Asn Pro Arg Gly Ser Ala 355360 365 Gly Pro Cys Cys Thr Pro Thr Lys Met Ser Pro Ile Asn Met Leu Tyr370 375 380 Phe Asn Asp Lys Gln Gln Ile Ile Tyr Gly Lys Ile Pro Gly MetVal 385 390 395 400 Val Asp Arg Cys Gly Cys Ser 405 3 630 DNA MouseGDF-11 CDS (198)..(575) 3 tctagatgtc aagagaagtg gtcacaatgt ctgggtgggagccgtaaaca agccaagagg 60 ttatggtttc tggtctgatg ctcctgttga gatcaggaaatgttcaggaa atcccctgtt 120 gagatgtagg aaagtaagag gtaagagaca ttgttgagggtcatgtcaca tctctttccc 180 ctctccctga ccctcag cat cct ttc atg gag ctt cgagtc cta gag aac 230 His Pro Phe Met Glu Leu Arg Val Leu Glu Asn 1 5 10acg aaa agg tcc cgg cgg aac cta ggc ctg gac tgc gat gaa cac tcg 278 ThrLys Arg Ser Arg Arg Asn Leu Gly Leu Asp Cys Asp Glu His Ser 15 20 25 agtgag tcc cgc tgc tgc cga tat cct ctc aca gtg gac ttt gag gct 326 Ser GluSer Arg Cys Cys Arg Tyr Pro Leu Thr Val Asp Phe Glu Ala 30 35 40 ttt ggctgg gac tgg atc atc gca cct aag cgc tac aag gcc aac tac 374 Phe Gly TrpAsp Trp Ile Ile Ala Pro Lys Arg Tyr Lys Ala Asn Tyr 45 50 55 tgc tcc ggccag tgc gaa tac atg ttc atg caa aag tat cca cac acc 422 Cys Ser Gly GlnCys Glu Tyr Met Phe Met Gln Lys Tyr Pro His Thr 60 65 70 75 cac ttg gtgcaa cag gcc aac cca aga ggc tct gct ggg ccc tgc tgc 470 His Leu Val GlnGln Ala Asn Pro Arg Gly Ser Ala Gly Pro Cys Cys 80 85 90 acc cct acc aagatg tcc cca atc aac atg ctc tac ttc aat gac aag 518 Thr Pro Thr Lys MetSer Pro Ile Asn Met Leu Tyr Phe Asn Asp Lys 95 100 105 cag cag att atctac ggc aag atc cct ggc atg gtg gtg gat cga tgt 566 Gln Gln Ile Ile TyrGly Lys Ile Pro Gly Met Val Val Asp Arg Cys 110 115 120 ggc tgc tcctaagttgtgg gctacagtgg atgcctccct cagaccctac 615 Gly Cys Ser 125cccaagaacc ccagc 630 4 126 PRT Mouse GDF-11 4 His Pro Phe Met Glu LeuArg Val Leu Glu Asn Thr Lys Arg Ser Arg 1 5 10 15 Arg Asn Leu Gly LeuAsp Cys Asp Glu His Ser Ser Glu Ser Arg Cys 20 25 30 Cys Arg Tyr Pro LeuThr Val Asp Phe Glu Ala Phe Gly Trp Asp Trp 35 40 45 Ile Ile Ala Pro LysArg Tyr Lys Ala Asn Tyr Cys Ser Gly Gln Cys 50 55 60 Glu Tyr Met Phe MetGln Lys Tyr Pro His Thr His Leu Val Gln Gln 65 70 75 80 Ala Asn Pro ArgGly Ser Ala Gly Pro Cys Cys Thr Pro Thr Lys Met 85 90 95 Ser Pro Ile AsnMet Leu Tyr Phe Asn Asp Lys Gln Gln Ile Ile Tyr 100 105 110 Gly Lys IlePro Gly Met Val Val Asp Arg Cys Gly Cys Ser 115 120 125 5 375 PRT HumanGDF-8 5 Met Gln Lys Leu Gln Leu Cys Val Tyr Ile Tyr Leu Phe Met Leu Ile1 5 10 15 Val Ala Gly Pro Val Asp Leu Asn Glu Asn Ser Glu Gln Lys GluAsn 20 25 30 Val Glu Lys Glu Gly Leu Cys Asn Ala Cys Thr Trp Arg Gln AsnThr 35 40 45 Lys Ser Ser Arg Ile Glu Ala Ile Lys Ile Gln Ile Leu Ser LysLeu 50 55 60 Arg Leu Glu Thr Ala Pro Asn Ile Ser Lys Asp Val Ile Arg GlnLeu 65 70 75 80 Leu Pro Lys Ala Pro Pro Leu Arg Glu Leu Ile Asp Gln TyrAsp Val 85 90 95 Gln Arg Asp Asp Ser Ser Asp Gly Ser Leu Glu Asp Asp AspTyr His 100 105 110 Ala Thr Thr Glu Thr Ile Ile Thr Met Pro Thr Glu SerAsp Phe Leu 115 120 125 Met Gln Val Asp Gly Lys Pro Lys Cys Cys Phe PheLys Phe Ser Ser 130 135 140 Lys Ile Gln Tyr Asn Lys Val Val Lys Ala GlnLeu Trp Ile Tyr Leu 145 150 155 160 Arg Pro Val Glu Thr Pro Thr Thr ValPhe Val Gln Ile Leu Arg Leu 165 170 175 Ile Lys Pro Met Lys Asp Gly ThrArg Tyr Thr Gly Ile Arg Ser Leu 180 185 190 Lys Leu Asp Met Asn Pro GlyThr Gly Ile Trp Gln Ser Ile Asp Val 195 200 205 Lys Thr Val Leu Gln AsnTrp Leu Lys Gln Pro Glu Ser Asn Leu Gly 210 215 220 Ile Glu Ile Lys AlaLeu Asp Glu Asn Gly His Asp Leu Ala Val Thr 225 230 235 240 Phe Pro GlyPro Gly Glu Asp Gly Leu Asn Pro Phe Leu Glu Val Lys 245 250 255 Val ThrAsp Thr Pro Lys Arg Ser Arg Arg Asp Phe Gly Leu Asp Cys 260 265 270 AspGlu His Ser Thr Glu Ser Arg Cys Cys Arg Tyr Pro Leu Thr Val 275 280 285Asp Phe Glu Ala Phe Gly Trp Asp Trp Ile Ile Ala Pro Lys Arg Tyr 290 295300 Lys Ala Asn Tyr Cys Ser Gly Glu Cys Glu Phe Val Phe Leu Gln Lys 305310 315 320 Tyr Pro His Thr His Leu Val His Gln Ala Asn Pro Arg Gly SerAla 325 330 335 Gly Pro Cys Cys Thr Pro Thr Lys Met Ser Pro Ile Asn MetLeu Tyr 340 345 350 Phe Asn Gly Lys Glu Gln Ile Ile Tyr Gly Lys Ile ProAla Met Val 355 360 365 Val Asp Arg Cys Gly Cys Ser 370 375 6 407 PRTHuman GDF-11 6 Met Val Leu Ala Ala Pro Leu Leu Leu Gly Phe Leu Leu LeuAla Leu 1 5 10 15 Glu Leu Arg Pro Arg Gly Glu Ala Ala Glu Gly Pro AlaAla Ala Ala 20 25 30 Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Val Gly GlyGlu Arg Ser 35 40 45 Ser Arg Pro Ala Pro Ser Val Ala Pro Glu Pro Asp GlyCys Pro Val 50 55 60 Cys Val Trp Arg Gln His Ser Arg Glu Leu Arg Leu GluSer Ile Lys 65 70 75 80 Ser Gln Ile Leu Ser Lys Leu Arg Leu Lys Glu AlaPro Asn Ile Ser 85 90 95 Arg Glu Val Val Lys Gln Leu Leu Pro Lys Ala ProPro Leu Gln Gln 100 105 110 Ile Leu Asp Leu His Asp Phe Gln Gly Asp AlaLeu Gln Pro Glu Asp 115 120 125 Phe Leu Glu Glu Asp Glu Tyr His Ala ThrThr Glu Thr Val Ile Ser 130 135 140 Met Ala Gln Glu Thr Asp Pro Ala ValGln Thr Asp Gly Ser Pro Leu 145 150 155 160 Cys Cys His Phe His Phe SerPro Lys Val Met Phe Thr Lys Val Leu 165 170 175 Lys Ala Gln Leu Trp ValTyr Leu Arg Pro Val Pro Arg Pro Ala Thr 180 185 190 Val Tyr Leu Gln IleLeu Arg Leu Lys Pro Leu Thr Gly Glu Gly Thr 195 200 205 Ala Gly Gly GlyGly Gly Gly Arg Arg His Ile Arg Ile Arg Ser Leu 210 215 220 Lys Ile GluLeu His Ser Arg Ser Gly His Trp Gln Ser Ile Asp Phe 225 230 235 240 LysGln Val Leu His Ser Trp Phe Arg Gln Pro Gln Ser Asn Trp Gly 245 250 255Ile Glu Ile Asn Ala Phe Asp Pro Ser Gly Thr Asp Leu Ala Val Thr 260 265270 Ser Leu Gly Pro Gly Ala Glu Gly Leu His Pro Phe Met Glu Leu Arg 275280 285 Val Leu Glu Asn Thr Lys Arg Ser Arg Arg Asn Leu Gly Leu Asp Cys290 295 300 Asp Glu His Ser Ser Glu Ser Arg Cys Cys Arg Tyr Pro Leu ThrVal 305 310 315 320 Asp Phe Glu Ala Phe Gly Trp Asp Trp Ile Ile Ala ProLys Arg Tyr 325 330 335 Lys Ala Asn Tyr Cys Ser Gly Gln Cys Glu Tyr MetPhe Met Gln Lys 340 345 350 Tyr Pro His Thr His Leu Val Gln Gln Ala AsnPro Arg Gly Ser Ala 355 360 365 Gly Pro Cys Cys Thr Pro Thr Lys Met SerPro Ile Asn Met Leu Tyr 370 375 380 Phe Asn Asp Lys Gln Gln Ile Ile TyrGly Lys Ile Pro Gly Met Val 385 390 395 400 Val Asp Arg Cys Gly Cys Ser405 7 23 DNA Artificial sequence Primer for PCR 7 gagtcccgct gctgccgatatcc 23 8 23 DNA Artificial sequence Primer for PCR 8 tagagcatgttgattgggga cat 23 9 20 DNA Artificial sequence Oligonucleotide probe forGDF-11 9 aaatatccgc atacccattt 20 10 376 PRT Mouse 10 Met Met Gln LysLeu Gln Met Tyr Val Tyr Ile Tyr Leu Phe Met Leu 1 5 10 15 Ile Ala AlaGly Pro Val Asp Leu Asn Glu Gly Ser Glu Arg Glu Glu 20 25 30 Asn Val GluLys Glu Gly Leu Cys Asn Ala Cys Ala Trp Arg Gln Asn 35 40 45 Thr Arg TyrSer Arg Ile Glu Ala Ile Lys Ile Gln Ile Leu Ser Lys 50 55 60 Leu Arg LeuGlu Thr Ala Pro Asn Ile Ser Lys Asp Ala Ile Arg Gln 65 70 75 80 Leu LeuPro Arg Ala Pro Pro Leu Arg Glu Leu Ile Asp Gln Tyr Asp 85 90 95 Val GlnArg Asp Asp Ser Ser Asp Gly Ser Leu Glu Asp Asp Asp Tyr 100 105 110 HisAla Thr Thr Glu Thr Ile Ile Thr Met Pro Thr Glu Ser Asp Phe 115 120 125Leu Met Gln Ala Asp Gly Lys Pro Lys Cys Cys Phe Phe Lys Phe Ser 130 135140 Ser Lys Ile Gln Tyr Asn Lys Val Val Lys Ala Gln Leu Trp Ile Tyr 145150 155 160 Leu Arg Pro Val Lys Thr Pro Thr Thr Val Phe Val Gln Ile LeuArg 165 170 175 Leu Ile Lys Pro Met Lys Asp Gly Thr Arg Tyr Thr Gly IleArg Ser 180 185 190 Leu Lys Leu Asp Met Ser Pro Gly Thr Gly Ile Trp GlnSer Ile Asp 195 200 205 Val Lys Thr Val Leu Gln Asn Trp Leu Lys Gln ProGlu Ser Asn Leu 210 215 220 Gly Ile Glu Ile Lys Ala Leu Asp Glu Asn GlyHis Asp Leu Ala Val 225 230 235 240 Thr Phe Pro Gly Pro Gly Glu Asp GlyLeu Asn Pro Phe Leu Glu Val 245 250 255 Lys Val Thr Asp Thr Pro Lys ArgSer Arg Arg Asp Phe Gly Leu Asp 260 265 270 Cys Asp Glu His Ser Thr GluSer Arg Cys Cys Arg Tyr Pro Leu Thr 275 280 285 Val Asp Phe Glu Ala PheGly Trp Asp Trp Ile Ile Ala Pro Lys Arg 290 295 300 Tyr Lys Ala Asn TyrCys Ser Gly Glu Cys Glu Phe Val Phe Leu Gln 305 310 315 320 Lys Tyr ProHis Thr His Leu Val His Gln Ala Asn Pro Arg Gly Ser 325 330 335 Ala GlyPro Cys Cys Thr Pro Thr Lys Met Ser Pro Ile Asn Met Leu 340 345 350 TyrPhe Asn Gly Lys Glu Gln Ile Ile Tyr Gly Lys Ile Pro Ala Met 355 360 365Val Val Asp Arg Cys Gly Cys Ser 370 375

1. A method of producing animal food products having an increased numberof ribs comprising: a) introducing a transgene disrupting or interferingwith expression of growth differentiation factor-11 (GDF-11) into anembryo into germ cells of a pronuclear embryo of the animal; b)implanting the embryo into the oviduct of a pseudopregnant femalethereby allowing the embryo to mature to full term progeny; c) testingthe progeny for presence of the transgene to identify transgene-positiveprogeny; d) cross-breeding transgene-positive progeny to obtain furthertransgene-positive progeny; and e) processing the progeny to obtainfoodstuff.
 2. The method of claim 1, wherein the transgene comprisesGDF-11 antisense polynucleotides.
 3. The method of claim 1, wherein thetransgene comprises a gene encoding a dominant negative GDF-11polypeptide.
 4. A method of producing avian, porcine or bovine foodproducts having an increased number of ribs comprising: a) introducing atransgene disrupting or interfering with expression of growthdifferentiation factor-11 (GDF-11) into an embryo of an avian, porcineor bovine animal; b) culturing the embryo under conditions wherebyprogeny are hatched; c) testing the progeny for presence of thetransgene to identify transgene-positive progeny; d) cross-breedingtransgene-positive progeny; and e) processing the progeny to obtainfoodstuff.
 5. The method of claim 4, wherein the transgene comprisesGDF-11 antisense polynucleotides.
 6. The method of claim 4, wherein thetransgene comprises a gene encoding a dominant negative GDF-11polypeptide.
 7. The transgenic animal of claim 4, wherein the transgenecomprises a polynucleotide encoding a truncated GDF-11 polypeptide.
 8. Amethod of treating a chronic or acute renal disease in a subject havingsuch a disease, comprising: administering to the subject, a reagentwhich affects GDF-11 activity or expression.
 9. The method of claim 8,wherein the reagent is an agonist of GDF-11.
 10. The method of claim 8,wherein the reagent is an antagonist of GDF-11.
 11. The method of claim10, wherein the antagonist is an antibody to GDF-11.
 12. The method ofclaim 10, wherein the antagonist is an antisense polynucleotide toGDF-11.